Investigation of the Effects of Blocking Potassium Channels With 4‐Aminopyridine on Paclitaxel Activity in Breast Cancer Cell Lines

Figures

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  FIGURE 1. Cell viability, colony formation and apoptotic cell number. Cell survival was determined by the trypan blue method on the hemocytometer. For 4‐ AP, the 4 mM concentration value was determined as IC50 and this concentration value was used in combination treatments for both cell lines. For PTX, 7.5 nM was found as IC50 for the MCF 7 cell line and 8 nM was found as IC50 for the MDA‐MB 231 cell line. A 7.5 nM concentration value was selected for the combination treatments. The survival comparison between separate drug treatments and combination treatment in the MCF‐7 cell line (A), and the MDA‐MB 231 cell line (B). Colony formation was performed after treatments, colony number decreased after combination treatment in MCF7 (C, E) and in MDA‐MB 231cell lines (D, F). Apoptosis determination was performed using Annexin/PI method by flow cytometer. Apoptotic cell numbers increased after combination treatment in MCF‐7 (G) and MDA‐MB‐231 cell lines (H). For all experiments, n ≥ 3, p < 0.0001 was indicated with **** (with respect to control).

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  FIGURE 2. The changes of intracellular K+ levels, intracellular Ca2+ concentration, and depolarization measurements of the transmembrane potential. After the treatments with 4‐AP, PTX, and the combination for 24 h, intracellular K+ was measured with PBFI/AM fluorescent dye by the fluorescence spectrophotometer. For all treatments, intracellular K+ levels increased with different ratios: MCF‐7 (A) and MDA‐MB‐231 (B) where the figures are represented with respect to control. After all treatments, determination of the intracellular Ca2+ was performed by Fura2 fluorescent dye. In MCF‐7 (E) and MDA‐MB‐231 (F) cell lines. To determine the depolarization of the transmembrane potential, a DiBAC4(3) fluorescent indicator was used. After all treatments, depolarization of the transmembrane potential was observed in the MCF‐7 cell line (C) and MDA‐MB‐231 (D) cell line. For all experiments, n ≥ 3, p < 0.05 was indicated *, p < 0.01 was indicated **, p < 0.001 was indicated ***, and p < 0.0001 was indicated with **** (with respect to control).

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  FIGURE 3. Determination of the cell cycle distribution. For detailed analyses, the cell cycle distribution was detected by a flow cytometer. In MCF‐7 cell line (A), the G1 arrest was observed after 4‐AP and PTX treatments. Additionally, PTX treatment caused a slight S‐phase arrest. The combination treatment caused the S phase arrest. In MDA‐MB‐231 cell line (B), 4‐AP caused G1 arrest, PTX caused S phase arrest, and the combination caused S phase and G2 arrest in MDA‐MB‐231 cell line. Determination of the cell cycle was also performed using a cell cycle assay kit by spectrophotometer. In MCF7 cell line (C), after 4‐AP, PTX, and the combination treatments, Cdk2 and Histone3 levels were determined by a cell cycle analysis kit. 4‐AP and PTX treatments caused an increase in Cdk2 levels. In Histone3 levels, there was no increase after all treatments [28]. In MDA‐MB 231 cell line (D), in Cdk2 level, 4‐AP caused an obvious increase, PTX caused a slight increase, and the combination treatment did not cause any change, in histone H3 levels. For all experiments, n ≥ 3, p < 0.05 was indicated *, p < 0.01 was indicated **, p < 0.001 was indicated *** (with respect to control).