Clotrimazole induces a late G sub(1) cell cycle arrest and sensitizes glioblastoma cells to radiation in vitro

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 G sub(1) cell cycle phase. Herein, we present data to support our hypothesis that clotrimazole arrests tumor cells in a radiosensitizing, late G sub(1) 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 G sub(1) 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 G sub(1) cell cycle phase. Concurrent with the late G sub(1) arrest, we observed an overexpression of p27 super(Kip) along with a decreased expression of p21 super(Cip), 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 18h 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 G sub(1) phase. The use of clotrimazole as a radiosensitizing agent for cancer treatment is novel and may have broad therapeutic applications.