Antitumor activity of the protein kinase inhibitor 1-(β-D-2'-deoxyribofuranosyl)-4,5,6,7-tetrabromo- 1H-benzimidazole in breast cancer cell lines

Abstract

Background: The protein kinases CK2 and PIM-1 are involved in cell proliferation and survival, the cell cycle, and drug resistance, and they are found overexpressed in virtually all types of human cancer, including breast cancer. In this study, we investigated the antitumor activity of a deoxynucleoside derivative, the protein kinase inhibitor compound 1-(β-D-2'-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole (K164, also termed TDB), inter alia CK2 and PIM-1, on breast cancer cell lines (MDA-MB-231, MCF-7, and SK-BR-3).

Methods: An evaluation of the cytotoxic and proapoptotic effects, mitochondrial membrane potential (ΔΨm), and cell cycle progression was performed using an MTT assay, flow cytometry, and microscopic analysis. The Western blotting method was used to analyze the level of proteins important for the survival of breast cancer cells and proteins phosphorylated by the CK2 and PIM-1 kinases.

Results: The examined compound demonstrated the inhibition of cell viability in all the tested cell lines and apoptotic activity, especially in the MCF-7 and SK-BR-3 cells. Changes in the mitochondrial membrane potential (ΔΨm), cell cycle progression, and the level of the proteins studied were also observed.

Conclusions: The investigated CK2 and PIM-1 kinase inhibitor K164 is a promising compound that can be considered a potential agent in targeted therapy in selected types of breast cancer; therefore, further research is necessary.

Keywords: 1-(β-D-2′-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole; Apoptosis; Breast cancer cell lines; Flow cytometry; Protein kinase inhibitor.

Fig. 1

The formula of the studied inhibitor 1-(β-D-2′-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole (K164, also termed TDB)

Fig. 2

The morphology of breast cancer cells cultured for 48 h, inverted microscopy. Panel A Control cells. Panel B Cells treated with compound (10 μM)

Fig. 3

Induction of apoptosis (sum of early and late apoptosis) by the inhibitor in BC cells. The data were determined by a FACS cytometer after 24 and 48 h of treatment. Cells were stained with Annexin V-FITC and PI. Each point represents the mean ± S.D.; asterisks indicate significance at p < 0.05 for comparison with the control

Fig. 4

Representative flow cytograms demonstrating changes in mitochondrial membrane potential (ΔΨm) of cells incubated with the K164 for 48 h. The cells were stained with JC-1 dye. The cells in the lower right region (R3) showed increased green fluorescence (apoptotic cells). Asterisks indicate significance at p < 0.05 for comparison with the control

Fig. 5

Western blot analysis of proteins in whole cell extracts obtained from cells cultured in the presence of K164 (concentrations of 10 μM, 20 μM, and 50 μM) after 48 h of incubation. Preparation of cell extracts and protein detection are described in Materials and Methods. Densitometry analysis was used to quantify all bands, and their intensity was normalized with respect to β-actin. For cell lines cultured in the absence of K164, the ratio of the examined proteins to β-actin was assumed to be 1. Panel A: PARP, AKT1, p 53, and B-cell lymphoma 2 (BCL-2) family proteins (pro- and anti- apoptotic). Panel B: phosphorylated proteins

Fig. 6

Changes in cell cycle progression in cells after 48 h treatment with K164. Each bar represents the mean ± S.D. (n ≥ 5). The data obtained from FACS Canto II flow cytometer (BD Biosciences, San Jose CA, USA) were analyzed using MacCycle software to determine the percentage of cells in each phase of the cell cycle. Each bar represents the mean ± SD; asterisks indicate significance at p < 0.05 for comparison with the control

Fig. 7

Exemplary DNA histograms of cell lines treated for 48 h with K164 compound (stained with propidium iodide)