Clotrimazole induces a late G1 cell cycle arrest and sensitizes glioblastoma cells to radiation in vitro

Abstract

Tumor cells are characterized by their high rate of glycolysis and clotrimazole has been shown to disrupt the glycolysis pathway thereby arresting the cells in the G1 cell cycle phase. Herein, we present data to support our hypothesis that clotrimazole arrests tumor cells in a radiosensitizing, late G1 phase. The effects of clotrimazole were studied using the glioblastoma cell line, U-87 MG. Flow cytometry was used to analyze cell cycle redistribution and induction of apoptosis. Immunoblots were probed to characterize a late G1 cell cycle arrest. Nuclear and cytoplasmic fractions were collected to follow the clotrimazole-induced translocation of hexokinase II. Clonogenic assays were designed to determine the radiosensitizing effect by clotrimazole. Our studies have shown a dose-dependent and time-dependent clotrimazole arrest in a late G1 cell cycle phase. Concurrent with the late G1 arrest, we observed an overexpression of p27 along with a decreased expression of p21, cyclin-dependent kinase 1, cyclin-dependent kinase 4, and cyclin D. Clotrimazole induced the translocation of mitochondrial-bound hexokinase II to the cytoplasm and the release of cytochrome c into the cytoplasm. Clotrimazole-induced apoptosis was enhanced when combined with radiation. Clotrimazole was shown to sensitize tumor cells to radiation when the cells were irradiated for 18 h post-clotrimazole treatment. The disruption of the glycolysis pathway by clotrimazole leads to cell cycle arrest of U-87 MG cells in the radiosensitizing late G1 phase. The use of clotrimazole as a radiosensitizing agent for cancer treatment is novel and may have broad therapeutic applications.

Fig. 1

Redistribution of the cell cycle by clotrimzole (CLT). U-87 MG cells were treated with CLT for various time points and processed for cell cycle analysis. DNA histograms corresponding to (a) G₁ phase, (b) S phase, and (c) G₂/M phase populations were expressed as a percentage of the total area of the DNA. The data represents the average ± standard error from three independent experiments, with each independent experiment done in triplicate. DMSO, dimethyl sulfoxide.

Fig. 2

The modulation of the expression of cell cycle regulatory proteins in U-87 MG cells after exposure to 40 μmol/l clotrimzole (CLT) or dimethyl sulfoxide (DMSO) treated control cells. Total cell lysates were collected and equal concentrations of protein (15 μg) was separated and electrophorectically blotted. Immunoblot analysis was used to follow the expression of cyclin D, cyclin-dependent kinase 1 (cdk1), cdk4, p27^Kip, and p21^Cip over a 30 h exposure time period. Actin was used as a loading control. Densitometry was used to quantitate the expression levels of each protein (data not shown). The representative result of one immunoblot is shown from three independent experiments.

Fig. 3

The modulation of the retinoblastoma protein (pRb) and the phosphorylation of pRb in U-87 MG cells after exposure to 40 μmol/l clotrimzole (CLT) or dimethyl sulfoxide (DMSO)-treated control cells. Total cell lysates were collected and equal concentrations of protein (15 μg) was separated and electrophorectically blotted. Phospho-Ser 807/811, phospho-Ser 780, and total pRb protein levels were detected by immunoblot during the treatment time period. Actin was used as a loading control. Densitometry was used to quantify the expression levels of each protein over the 30 h time period. The representative result of one immunoblot is shown from three independent experiments.

Fig. 4

Immunoblot analysis for the translocation of hexokinase II (HKII) and the release of cytochrome c (Cyto C) from the mitochondria to the cytoplasm after U-87 MG cells were exposed to 40 μmol/l clotrimzole (CLT) or dimethyl sulfoxide (DMSO) for 6 h. Mitochondrial and cytoplasmic fractions were isolated and equal protein concentrations (20 μg) were separated and electrophorectically blotted. The immunoblots were probed for HKII and Cyto C to observe the effect by CLT on the translocation of HKII and the release of Cyto C. Actin was used as a loading control. Densitometry was used to quantify the expression levels of each protein in the cytoplasmic and mitochondrial cell fractions. The representative result of one immunoblot is shown from two independent experiments.

Fig. 5

Detection of apoptosis in U-87 MG cells treated with clotrimzole (CLT) for 24 h, followed by radiation treatment (RT) (3 Gy). Cells were collected and processed immediately after 3 Gy RT (24 h time point), or 48 and 72 h after treatment with CLT. Data points represent the average ± standard error of three independent experiments, with each independent experiment done in triplicate. A significant increase in the detection of apoptotic cells after RT as compared with CLT treated cells was observed at 24 h (*P=0.021) and 72 h (**P=0.048).

Fig. 6

Cell survival curves for the detection of radiosensitization by clotrimzole (CLT). U-87 MG cells were treated with 5 μmol/l CLT (◇), 10 μmol/l CLT (△), 20 μmol/l CLT (○) or vehicle [dimethyl sulfoxide (DMSO), □] for 18 h before the treatment with radiation. After an incubation period of 12–15 days, the colonies were fixed, stained and counted, analyzed, and graphed (surviving fraction vs. radiation dose). The average plating efficiencies were 43% (DMSO), 34% (5 μmol/l CLT), 21% (10 μmol/l CLT), and 5% (20 μmol/l CLT). A significant radiosensitizing effect by CLT was observed at 5 μmol/l (*P= 0.0455), 10 μmol/l (**P= 0.0040), and 20 μumol/l (***P= 0.0019) as compared with the DMSO control group. All data are the combined results from three independent experiments, with each independent experiment done in triplicate.

Fig. 7

A schematic representation of the anticancer activity of clotrimazole using data presented herein and earlier published. The mitochondrial-bound hexokinase II enzyme converts glucose to glucose-6-phosphate in the first step of the glycolysis pathway. Clotrimazole disrupts the glycolysis pathway by the translocation of hexokinase II from the voltage-dependent anion channel on the outer mitochondrial membrane to the cytoplasm [4,43,44]. Bax is now capable of binding to the voltage-dependent anion channel and inducing the release of cytochrome c into the cytoplasm, thereby inducing apoptosis [18]. The translocation of hexokinase II interferes with the glycolysis pathway and reduces ATP production [45]. The cell responds by arresting in late G₁, thereby preventing the cells from progressing into the DNA synthesis phase [39]. The arrested cells are sensitive to radiation and cisplatin treatments, with an enhancement of apoptosis [9].