Synergistic Interactions of 5-Fluorouracil with Inhibitors of Protein Kinase CK2 Correlate with p38 MAPK Activation and FAK Inhibition in the Triple-Negative Breast Cancer Cell Line

Abstract

Background: The combination effect of 5-fluorouracil (5-FU) with either CX-4945 or a new inhibitor of protein kinase CK2, namely 14B (4,5,6,7-tetrabromo-1-(3-bromopropyl)-2-methyl-1H-benzimidazole), on the viability of MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines was studied. Methods: Combination index (CI) values were determined using an MTT-based assay and the Chou-Talalay model. The effect of the tested drug combinations on pro-apoptotic properties and cell cycle progression was examined using flow cytometry. The activation of FAK, p38 MAPK, and ERK1/2 kinases and the expression of selected pro-apoptotic markers in MDA-MB-231 cell line after the combined treatment were evaluated by the western blot method. Confocal microscopy was used to examine actin network in MDA-MB-231. Results: Our results showed that a synergistic effect (CI < 1) occurred in MDA-MB-231 after treatment with both combinations of 5-FU with 14B or CX-4945, whereas the combination of 5-FU and 14B evoked an antagonistic effect in MCF-7. We conclude that the synergistic interactions (CI < 1) observed for both the combinations of 5-FU and 14B or CX-4945 in MDA-MB-231 correlated with an activation of p38 MAPK, inhibition of FAK, increased expression of apoptogenic markers, prolongation of S-phase of cell cycle, and destabilization of actin network. Conclusions: The obtained results support the recent observation that CK2 inhibitors can improve 5-FU-based anticancer therapy and FAK kinase can be an attractive molecular target in breast cancer therapy.

Inhibition of Protein Kinase CK2 Affects Thymidylate Synthesis Cycle Enzyme Level and Distribution in Human Cancer Cells

“…3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based viability assay: after incubation with the test compounds, an MTT test was performed as described previously (Wińska et al, 2020). Optical densities were measured at 570 nm using a BioTek microplate reader.…”

“…After incubation, the suspension cells were collected by centrifugation at 260 RCF and washed 3 times with ice-cold PBS and supernatants were discarded and pellets were storage at -20 °C up to 1 month. In order to obtain lysates, RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 0.2 mM sodium orthovanadate, and protease inhibitors cocktail, Roche) was added as it was described elsewhere (Wińska et al, 2020). Adherent monolayer cells were washed three times in ice-cold PBS, and the cells were scraped and lysed in RIPA as it was described previously (Wińska et al, 2020).…”

“…In order to obtain lysates, RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 0.2 mM sodium orthovanadate, and protease inhibitors cocktail, Roche) was added as it was described elsewhere (Wińska et al, 2020). Adherent monolayer cells were washed three times in ice-cold PBS, and the cells were scraped and lysed in RIPA as it was described previously (Wińska et al, 2020). Cellular lysates were analyzed by western blotting as it was described previously (Wińska et al, 2019).…”

“…Taking into account that CK2 regulates many oncogenic pathways and processes, we have recently undertaken the studies on simultaneous treatment of cancer cells with inhibitors of TS and CK2 (Wińska et al, 2018;Wińska et al, 2020) or DHFR and CK2 (Wińska et al, 2019) searching for the synergistic effect. The obtained results demonstrate the ability of CK2 inhibitors to enhance the efficacy of 5-fluorouracil (FU, TS inhibitor) or methotrexate (MTX, DHFR inhibitor) in cancer cells.…”

Protein Kinases: Function, Substrates, and Implication in Diseases

“…Concerning the original research articles, Wińska et al tested the combination effect of 5-fluorouracil (5-FU), with specific inhibitors of protein kinase CK2 in the triple-negative breast cancer cell line. They found that inhibitors of protein kinase CK2 can improve 5-FU-based anticancer therapy and proposed FAK kinase as an attractive target for cancer therapy [ 8 ]. In the next research article, Szoltysek et al focused on DRAK2, a novel stress response kinase that plays a critical role in apoptosis, T-cell biology, and B-cell activation in chronic lymphocytic leukemia (CLL).…”