|Year : 2020 | Volume
| Issue : 4 | Page : 163-168
Anticancer evaluation of N-benzoyl-3-allylthiourea as potential antibreast cancer agent through enhances HER-2 expression
Tri Widiandani1, Siswandono1, Edy Meiyanto2
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Airlangga, Surabaya 60155, Indonesia
2 Department of Pharmaceutical Chemistry; Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
|Date of Submission||09-Jun-2020|
|Date of Decision||30-Jun-2020|
|Date of Acceptance||20-Aug-2020|
|Date of Web Publication||10-Oct-2020|
Dr. Tri Widiandani
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Airlangga, Surabaya 60155; Gedung Nanizar Zaman Joenoes. Kampus C UNAIR, Jl Mulyorejo, Surabaya
Source of Support: None, Conflict of Interest: None
Breast cancer with HER-2 overexpression is sensitive to drugs which target the receptor or its kinase activity. Although the anti-HER-2 therapies commonly used have improved patient outcome, resistance usually occurs. In this present study, we investigated a modification of the chemical structure of allylthiourea derivatives in order to enhance the cytotoxicity effect on breast cancer cells with HER-2 overexpression. The aim of this research was to predict the absorption, distribution, metabolism, excretion, and toxicity by in silico study and to explore the effect N-benzoyl-3-allylthiourea (BATU) on MCF-7 cell line with overexpressing of HER-2 using MTT assay and western blot. The result showed that the cytotoxicity effects of BATU on MCF-7/HER-2 cell line (IC50value 0.64 mM) were higher than on MCF-7 cell lines (IC50value 1.47 mM). In addition, the cytotoxic effects of BATU on MCF-7 and MCF-7/HER-2 were higher than allylthiourea as a lead compound (IC50value 5.22 and 3.17 mM). The results also confirmed that the BATU compound has the ability to effectively enhance its cytotoxicity against MCF-7/HER-2 through enhanced HER-2 expression and inhibition of nuclear factor kappa B (NF-kB) activation. Above all, the BATU compound is effective in increasing HER-2 expression and inactivating NF-kB transcription factors, thereby resulting in inhibition of protein expression which works a significant part in cell proliferation. Therefore, the BATU compound has the potential to be developed as a complementary drug in breast cancer therapy with HER-2 positive.
Keywords: Allylthiourea, cytotoxicity, HER-2, MCF-7, N-benzoyl-3-allylthiourea, nuclear factor kappa B
|How to cite this article:|
Widiandani T, Siswandono, Meiyanto E. Anticancer evaluation of N-benzoyl-3-allylthiourea as potential antibreast cancer agent through enhances HER-2 expression. J Adv Pharm Technol Res 2020;11:163-8
|How to cite this URL:|
Widiandani T, Siswandono, Meiyanto E. Anticancer evaluation of N-benzoyl-3-allylthiourea as potential antibreast cancer agent through enhances HER-2 expression. J Adv Pharm Technol Res [serial online] 2020 [cited 2021 Oct 26];11:163-8. Available from: https://www.japtr.org/text.asp?2020/11/4/163/297698
| Introduction|| |
Based on the World Health Organization in 2018, a total of 627,000 deaths are caused by breast cancer, the highest contributor to death from all types of cancer in women. In cases of breast cancer, 30% of occurrences are due to the overexpression of HER-2 with a poor prognosis., A large number of HER-2 receptors are able to influence the continuous proliferation of tumor cells and induce spontaneous dimerization and autophosphorylation, trigger the activation of focal adhesion kinase, and induce migration processes and cancer cell metastasis.
The most common treatment used for breast cancer cases with HER-2 is the lapatinib chemotherapy agent. According to Johnston et al. (2006), lapatinib plays as a tyrosine kinase inhibitor. However, the use of lapatinib has been reported to be resistant to HER-2 positive breast cancer cases. Other anticancer group such as the thiourea derivative, which acts as an inhibitor of epidermal growth factor receptor (EGFR) kinase, causes high antiproliferation activity against tumor cells. In addition, these compounds play a significant role in inhibiting the protein tyrosine kinase and NADH oxidase, which both contribute to the growth of tumor cells.,
In recent years, several studies were conducted on the development of thiourea derivatives as anticancer agents. The results showed that the compound developed had a pharmacophore group thiourea (-HN-C (=S)-NH-).,,,, The synthesis and relationship of the structural activity of N-benzyl-N-(X-2-hydroxybenzyl)-N-phenylurea and thiourea derivatives as anticancer showed that the derivatives of these compounds work as potential inhibitors of EGFR and HER-2 and have a high antiproliferation activity against MCF-7. Furthermore, recent studies on allylthiourea derivatives showed the activity of these compounds against T47D and MCF-7 line breast cancer cells., In this study, the activity of allylthiourea derivatives, namely N-benzoyl-3-allylthiourea (BATU) [Figure 1], will be tested for the anticancer activity using MCF-7 cells with HER2 overexpression.
|Figure 1: The route structure of N-benzoyl-3-allylthiourea. Allylthiourea (a) reacted with benzoyl chloride in an alkaline state will produce the target compound N-benzoyl-3-allylthiourea (b)|
Click here to view
| Materials and Methods|| |
Absorption, distribution, metabolism, excretion, and toxicity prediction
The prediction of pharmacokinetic properties including absorption, distribution, metabolism, excretion, and toxicity (ADMET) of BATU compounds was carried out using the online pkCSM tool program.,, These BATU and comparative compounds were drawn in 2D and 3D molecular structures with ChemBioOffice Ultra 13.0 programs, which were subsequently stored as *.sdf files. Then, the structures of the BATU compound were translated into the SMILES format using the Online SMILES Translator program. In the SMILES format, these compounds were processed using the online pkCSM tool to predict ADMET of the compounds.
The silico test involved the use of human EGFR with ID code 3PP0 downloaded from Protein Data Bank (PDB). This protein contains the standard ligand SYR127063. The work procedure of the in silic o test was conducted in several stages, starting with the preparation of the test ligand used to make a 2D and 3D structure of the compound through the ChemBioOffice Ultra 13.0 programs. The next step was energy minimization with MMFF94. Then, the structures were stored with the extension * mol2/SYBYL2. Then, the Molegro Virtual Docker 5.5 program is used to carry out the molecular docking process.,,
The test subjects used were MCF-7/HER-2 and MCF-7 cells, collected from the Nara Institute of Science and Technology, Japan. The cell cultures were grown in Dulbecco's Modified Eagle Medium (DMEM) media containing FBS 10% (v/v) from Sigma and 1% (v/v) penstrep antibiotics from Nacalai Tesque. Then, 0.25% trypsin-EDTA from Nacalai Tesque was used for cell harvesting.
The MCF-7/HER-2 and MCF-7 cells were distributed into 96 well plates and incubated in a CO2 incubator for 24 h. After which, the test solution was added in various concentration series and re-incubated for another 24 h. After that, each plate was added 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in PBS from Sigma. The incubation continued for 4 h at 37°C until formazan was formed. The MTT reaction was stopped with 10% SDS from Nacalai Tesque in 0.01 N HCl from Merck, after which the incubation was allowed to continue overnight at room temperature. The uptake was read by ELISA reader at a wavelength of 570 nm and the absorbance results read were converted into the percentage of life.,
In this method, 8 × 105 cells were planting in a 6 well plate and incubated for 24 h then treated with the sample for 24 h. After which, the cells were lysed using lysis buffer consisting of 1% NP40, 5 mM EDTA in Tris buffer, for 30 min. The cell suspension was centrifuged at 15,000 rpm for 20 min. The SDS-polysaccharide gel was then electrophoresed with a current of 30–40 mA for approximately 1 h. Then, the protein was transferred to the Polyvinylidene difluoride (PVDF) membrane using a blotting machine for 1–2 h. Next, the PVDF membrane was blocked with the use of blocking buffer for 1 h, after which it was incubated with first antibodies which was anti-HER-2 at 1:50 dilution and anti-p65 at 1:300 dilution. The protein at that point formed hybridization with the first antibodies and then incubated with second antibodies known as antimouse IgG, at 1: 2000 dilution. The protein in the membrane was detected with the use of Chemilumi-one plus.
| Results|| |
Absorption, distribution, metabolism, excretion and toxicity prediction
The predicted results of the pharmacokinetic properties of ADMET from the BATU and allylthiourea (ATU) compounds as well as the comparative hydroxyurea (HU) and lapatinib compounds are shown in [Table 1].
|Table 1: Predictions of absorption, distribution, metabolism, excretion, and toxicity|
Click here to view
A description of the interaction of amino acid residues from the BATU compound at the pdb code HER-2 receptor 3PP0 and the acquisition of the docking value (Rerank Score [RS]) is shown in [Figure 2].
|Figure 2: (A) Interactions between compounds (a) N-benzoyl-3-allylthiourea, (b) SYR127063, (c) allylthiourea and (d) lapatinib with amino acids at HER-2 receptors with using MVD programs are shown in 3D. In the picture, the blue lines show interactions in hydrogen bonds. Tests carried out on human epidermal growth factor-2, pdb codes 3PP0 by calculating the RMSD factor. (B) The graphs of in silico test results indicated by the Rerank score parameter values, from N-benzoyl-3-allylthiourea, allylthiourea, hydroxyurea, and LP, which are compared with the standard ligand SYR127063. The more negative value indicates the more stable interaction energy|
Click here to view
Based on the docking result, it can be known that BATU has binding energy with receptors which is reflected by the RS is lower than ATU. The BATU derivative also shows a lower RS than hydroxyurea.
IC50 values of BATU compounds against MCF-7 and MCF7/HER2 cells by MTT method and amino acid residues that interact with BATU compounds are shown in [Table 2]. The results of cytotoxic tests on MCF-7 and MCF7/HER-2 breast cancer cells are shown in [Figure 3].
|Figure 3: (A) Effect of sample treatment of N-benzoyl-3-allylthiourea compound on the morphology of MCF7 and MCF-7/HER-2 cells after incubation for 24 h. Observation of cell morphology at 24 h was carried out with an inverted microscope at a magnification of ×100. Blue lines indicate dead cells. (B) Percentage of living cells (cell viability) curve after treatment of test compounds in different concentrations of (a) MCF-7 and (b) MCF-7/HER-2 cells of N-benzoyl-3-allylthiourea, allylthiourea, and hydroxyurea compounds|
Click here to view
The result of cytotoxicity showed that the treatment of BATU compounds caused changes in morphology and cell density of MCF-7/HER-2 along with an increase in test concentration. MCF-7/HER-2 breast cancer cells in control are wide in shape with clear cytosol, colonized, and attached to the base of the tissue culture dish. After the treatment of BATU compounds, some cells appear to be smaller and detached from TCD (a). The morphological changes of MCF-7/HER-2 cells are in line with the increase in the concentration of the test compound (b).
HER-2 protein expression and p-65 localization
The expression of HER-2 due to the treatment of BATU and ATU compounds at various concentrations of MCF-7/HER-2 cells and localization of p-65 transcription factor (nuclear factor kappa B [NF-kB]) by western blot method are shown in [Figure 4].
|Figure 4: (a) (i) HER-2 expression of test compounds at various concentrations to MCF-7/HER-2 cells. Cells without treatment (control) and cells with treatment (N-benzoyl-3-allylthiourea and allylthiourea) with concentrations of 100, 250, 500, 750, and 1000 uM for 24 h, analyzed by western blot method. (ii) Analysis of the results was carried out using ImageJ® software to measure the intensity of the HER-2 expression band on the MCF-7/HER-2 cell line compared to the control. (b) Expression and localization of p65 (nuclear factor kappa B) due to the treatment of N-benzoyl-3-allylthiourea samples on MCF-7/HER-2 cells for 24 h by western blot method at N-benzoyl-3-allylthiourea concentrations of 750 uM and 1000 uM|
Click here to view
The results of western blot analysis showed an increase in HER-2 protein expression along with an increase in the concentration of BATU compounds. At the lowest concentration (100 μM) did not show a different expression intensity compared to the control, and the intensity of expression increases with increasing concentration of BATU compounds.
| Discussion|| |
The prediction results of the pharmacokinetic properties of ADMET [Table 1] show that the BATU compound has good skin permeability (log Kp <-2.5). Similarly, for the prediction of distribution volume, the BATU compound has a log value of steady state of volume distribution (VDss) >-0.15, hence it has a fairly good distribution volume. However, the comparative compound has a low distribution volume since it has a log value of VD <-0.15. Additionally, the ability of BATU compounds to penetrate the blood–brain barrier (BBB) is quite low because it has a log value of BBB>-1. Therefore, there is a need for the consideration of the drugs' ability to penetrate the BBB in order to increase the efficacy of drugs whose pharmacological activities are in the brain. In addition, the ability of the compound to inhibit cytochrome P450 was shown by cytochrome P2D6 isoform (CYP2D6). Hence, from [Table 1], it is shown that the BATU compound does not affect cytochrome P450. Furthermore, organic cation transporter 2 (OCT2) is a transporter in the kidneys which works an important part in the disposition and clearance of drugs as well as endogenous compounds. It is seen that all BATU do not affect the OCT2 substrate. Then, the toxicity of compounds was determined by the Ames toxicity test. This test assesses the mutagenic potential of a compound with the use of bacteria. From [Table 1], it can be determined that BATU compounds and comparators do not have mutagenic and nonhepatotoxic potential.
Furthermore, the BATU compound has a binding energy with the receptor as shown in [Table 2], reflected by a lower RS compared to ATU. The BATU compounds also show a lower RS compared with HU, considering the fact that BATU derivatives have advantages over ATU and HU, and with the ability to bind with the amino acids Met801 and Thr 862 through hydrogen bonds, and amino acids Gly804, Thr798, Gln799, Ala751, Ile752, Val797, Leu796 through steric bonds as shown in [Figure 2]. Furthermore, BATU compounds interact with amino acid residues at the same ATP binding site as standard ligands, namely Thr862 and Met801 through hydrogen bonds. The results of this molecular modeling study showed that the BATU compound has the potential to be developed with the prediction of having higher biological activity compared with ATU and HU.
The results also show the IC50 value of BATU compound was smaller than ATU on MCF-7 and MCF-7/HER-2 cell line. This is an indication that the BATU compound has a higher cytotoxic effect on breast cancer cell compared with the ATU. Besides, the cytotoxic test also shows that the BATU compound has a higher activity against the MCF-7/HER-2 than on the MCF-7 cell line. Hence, it is suspected that the compound works more selectively against MCF-7 cells which are overexpressed by HER-2. The results of this cytotoxic test were strengthened by an analysis of HER-2 protein expression using the western blot method. The results of this western blot analysis show that the BATU compound exhibits expression activity on HER-2 protein.
It is also clear from the results from this study that the higher the concentration of the BATU compound, the higher the expression of HER-2 protein in MCF-7/HER-2 cell line. This study shows that the localization of p65 in the cytoplasm and inactivation of p65 in the cell nucleus were overexpressed by BATU compounds, with concentrations around IC50 values or higher concentrations of about 1000 μM. Therefore, this has proven the fact that BATU compound has the ability to effectively inhibit the activation of NF-kB.
Based on these results, there is need to do further research on BATU compound developed as a potential therapy drug and for the reduction of the occurrence of resistance to anticancer drugs for breast cancer with HER-2 expression. Moreover, the flow cytometry test can be proposed as a suggestion to be done in the next research to check if BATU could engage cell cycle profile.
| Conclusion|| |
Based on this study, it can be concluded that the BATU compound is effective in increasing HER-2 expression and inactivating NF-kB transcription factors, thereby resulting in inhibition of protein expression which works a significant part in cell proliferation. Therefore, the BATU compound has the potential to be developed as a complementary drug in breast cancer therapy with HER-2 positive.
This research was supported by the Ministry of Research Technology and Higher Education, The Republic of Indonesia. We are also very grateful to Professor Yasumasha Bessho and Assoc. Professor Norihiro Ishida-Kitagawa for the facilities at the Laboratory of Gene Regulation, Department of Biological Science, Nara Institute of Science and Technology, Japan.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Gibbs JB. Anticancer drug targets: Growth factors and growth factor signaling. J Clin Invest 2000;105:9-13.
Laskin JJ, Sandler AB. Epidermal growth factor receptor: A promising target in solid tumours. Cancer Treat Rev 2004;30:1-7.
Johnson E, Seachrist DD, DeLeon-Rodriguez CM, Lozada KL, Miedler J, Abdul-Karim FW, et al
. HER2/ErbB2-induced breast cancer cell migration and invasion require p120 catenin activation of Rac1 and Cdc42. J Biol Chem 2010;285:29491-501.
Johnston SR, Leary A. Lapatinib: A novel EGFR/HER2 tyrosine kinase inhibitor for cancer. Drugs Today (Barc) 2006;42:441-53.
Mitra D, Brumlik MJ, Okamgba SU, Zhu Y, Duplessis TT, Parvani JG, et al
. An oncogenic isoform of HER2 associated with locally disseminated breast cancer and trastuzumab resistance. Mol Cancer Ther 2009;8:2152-62.
Yang W, Hu Y, Yang YS, Zhang F, Zhang YB, Wang XL, et al
. Design, modification and 3D QSAR studies of novel naphthalin-containing pyrazoline derivatives with/without thiourea skeleton as anticancer agents. Bioorg Med Chem 2013;21:1050-63.
Li HQ, Yan T, Ying Y, Shi L, Zhou CF, Zu HL. Synthesis and structure-activity relationship of N-benzyl-N-(X-2-hydroxybenzyl)-N'-phenylureas and thioureas as antitumor agents. Bioorg Med Chem 2010;18:305-13.
Manjula SN, Malleshappa Noolvi N, Vipan Parihar K, Manohara Reddy SA, Ramani V, Gadad AK, et al
. Synthesis and antitumor activity of optically active thiourea and their 2-aminobenzothiazole derivatives: A novel class of anticancer agents. Eur J Med Chem 2009;44:2923-9.
Thanigaimalai P, Lee KC, Sharma VK, Joo C, Cho WJ, Roh E, et al
. Structural requirement of phenylthiourea analogs for their inhibitory activity of melanogenesis and tyrosinase. Bioorg Med Chem Lett 2011;21:6824-8.
McCarthy AR, Sachweh MC, Higgins M, Campbell J, Drummord CJ, Leeuwen IM, et al.
Tenovin-D3, a novel small-molecule inhibitor of sirtuin SirT2, increases p21 (CDKN1A) expression in a p53-independent manner. Mol Cancer Ther 2013;12:352–60.
Huang XC, Wang M, Pan YP, Yao GY, Wang HS, Tiang XY, et al
. Synthesis and antitumor activities of novel thiourea α-aminophosphonates from dehydroabietic acid. Eur J Med Chem 2013;69:508-20.
Koca İ, Özgür A, Coşkun KA, Tutar Y. Synthesis and anticancer activity of acyl thioureas bearing pyrazole moiety. Bioorg Med Chem 2013;21:3859-65.
Widiandani T, Arifianti L, Siswandono. Docking, synthesis, and cytotoxicity test on human breast cancer cell line (t47d) of N-(Allylcarbamothioyl) benzamide. Int J Pharm Clin Res 2016;8:372-6.
Widiandani T, Siswandono S, Meiyanto E, Sulistyowaty MI, Purwanto BT, Hardjono S. New N-Allylthiourea derivatives: Synthesis, molecular docking andin vitro
cytotoxicity studies. Trop J Pharm Res 2018;17:1607-13.
Pires DE, Blundell TL and Ascher DB. pkCSM: Predicting small molecule pharmacokinetic and toxicity properties using graph-based signatures. J Med Chem 2015;58:4066-72.
Saeed S, Rashid N, Jones PG, Ali M, Hussain R. Synthesis, characterization and biological evaluation of some thiourea derivatives bearing benzothiazole moiety as potential antimicrobial and anticancer agents. Eur J Med Chem 2010;45:1323-31.
Hardjono S, Widiandani T, Purwanto BT, Nasyanka AL. Molecular docking of N-benzoyl-N'-(4-fluorophenyl) thiourea derivatives as anticancer drug candidate and their ADMET prediction. Res J Pharm Tech 2019;12:2160-6.
Cbio CL. Molegro Virtual Docker User Manual, MVD 2012.5.5 for Windows, Linux, and Mac OS X. Molegro – A CLC Bio Company; 2012.
Widiandani T, Siswandono. Structure modification and anticancer activity prediction of allylthiourea derivatives base on In silico
. e-J BIKFar 2015;4:33-40.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]