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.