Hyperthermia sensitizes Rhizopus oryzae to posaconazole and itraconazole action through apoptosis

The high mortality rate of mucormycosis with currently available monotherapy has created interest in studying novel strategies for antifungal agents. With the exception of amphotericin B (AMB), the triazoles (posaconazole [PCZ] and itraconazole [ICZ]) are fungistatic in vitro against Rhizopus oryzae . We hypothesized that growth at a high temperature (42°C) results in fungicidal activity of PCZ and ICZ that is mediated through apoptosis. R. oryzae had high MIC values for PCZ and ICZ (16 to 64 μg/ml) at 25°C; in contrast, the MICs for PCZ and ICZ were significantly lower at 37°C (8 to 16 μg/ml) and 42°C (0.25 to 1 μg/ml). Furthermore, PCZ and ICZ dose-dependent inhibition of germination was more pronounced at 42°C than at 37°C. In addition, intracellular reactive oxygen species (ROS) increased significantly when fungi were exposed to antifungals at 42°C. Characteristic cellular changes of apoptosis in R. oryzae were induced by the accumulation of intracellular reactive oxygen species. Cells treated with PCZ or ICZ in combination with hyperthermia (42°C) exhibited characteristic markers of early apoptosis: phosphatidylserine externalization visualized by annexin V staining, membrane depolarization visualized by bis-[1,3-dibutylbarbituric acid] trimethine oxonol (DiBAC) staining, and increased metacaspase activity. Moreover, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and DAPI (4',6-diamidino-2-phenylindole) staining demonstrated DNA fragmentation and condensation, respectively. The addition of N-acetylcysteine increased fungal survival, prevented apoptosis, reduced ROS accumulation, and decreased metacaspase activation. We concluded that hyperthermia, either alone or in the presence of PCZ or ICZ, induces apoptosis in R. oryzae. Local thermal delivery could be a therapeutically useful adjunct strategy for these refractory infections.