Anticancer evaluation of N-benzoyl-3-allylthiourea as potential antibreast cancer agent through enhances HER-2 expression

Abstract

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 (IC₅₀ value 0.64 mM) were higher than on MCF-7 cell lines (IC₅₀ value 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 (IC₅₀ value 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; HER-2; MCF-7; N-benzoyl-3-allylthiourea; cytotoxicity; nuclear factor kappa B.

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)

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

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

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