Rotenone Enhances the Antifungal Properties of Staurosporine

Abstract

We studied staurosporine-induced cell death in the filamentous fungus Neurospora crassa. The generation of reactive oxygen species during the process appears to be an important signaling event, since addition of the antioxidant glutathione prevents the effects of staurosporine on fungal growth. Selected mutants with mutations in respiratory chain complex I are extremely sensitive to the drug, stressing the involvement of complex I in programmed cell death. Following this finding, we determined that the complex I-specific inhibitor rotenone used in combination with staurosporine results in a synergistic and specific antifungal activity, likely through a concerted action on intracellular glutathione depletion. Paradoxically, the synergistic antifungal activity of rotenone and staurosporine is observed in N. crassa complex I mutants and in Saccharomyces cerevisiae, which lacks complex I. In addition, it is not observed when other complex I inhibitors are used instead of rotenone. These results indicate that the rotenone effect is independent of complex I inhibition. The combination of rotenone and staurosporine is effective against N. crassa as well as against the common pathogens Aspergillus fumigatus and Candida albicans, pointing to its usefulness as an antifungal agent.Programmed cell death (PCD) refers to a genetically controlled process of cellular suicide initiated by endogenous or extrinsic signals. Many of the genes involved are widely conserved from unicellular to multicellular organisms (46). Apoptosis and autophagy, with its particular characteristics, have been recognized as the main categories of PCD (27). The process of PCD is crucial for the development and homeostasis of metazoan organisms and has been implicated in a number of human disorders, including cancer and neurodegenerative and infectious diseases (3,10,25,55).The participation in PCD of mitochondria, the cellular organelles responsible for the production of most cellular ATP in eukaryotes (30), has been well established. Particularly, these organelles have a central role in the intrinsic (mitochondriondependent) pathway of apoptosis, which includes production of reactive oxygen species (ROS), membrane depolarization, ultrastructural changes, and the release of cytochrome c and other proteins (18,50,55). Drugs like staurosporine (STS), an inhibitor of protein kinases, have been used to induce the mitochondrion-dependent pathway of apoptosis (24, 35). Staurosporine (48) and derivatives have been used in clinical trials for cancer therapy (63). The complex I inhibitor rotenone too has been widely used to induce PCD and also extensively applied as a pesticide (11,39,56). Thus, these types of drugs can be employed for the acquisition of fundamental knowledge and for more practical applications, like modulation of the progression of PCD.Modulation of PCD by targeting metabolic pathways involved in the process can be exploited to the benefit of human health in several very significant situations, from cancer therapy (4, 57) to the treatment of f...

Transcriptional analysis of the response of Neurospora crassa to phytosphingosine reveals links to mitochondrial function

“…In these experiments, the complex I mutant nuo14 and a mutant lacking an AMID-like protein (DNCU06061.2) were used as controls. The nuo14 mutant is more resistant to PHS, while DNCU06061.2 is more sensitive to PHS than the wild-type strain (Castro et al, 2008). Among the 24 mutants carrying deletions in genes whose expression is affected by PHS, we identified two strains that were more resistant to the drug than the wildtype strain (Fig.…”

“…The green and black colours indicate high and low expression levels, respectively. We compared the response of wild-type and a complex I mutant (nuo14) to treatment with H 2 O 2 ; the resistance of complex I mutants towards H 2 O 2 is more marked than their resistance towards PHS (Castro et al, 2008). Our experimental design is illustrated in Fig.…”

“…PHS induces rapid PCD in N. crassa (Castro et al, 2008). In order to analyse the transcriptional response to this drug, we set up the time-course experiment depicted in Fig.…”

“…We use the filamentous fungus Neurospora crassa as a model organism because it offers many experimental advantages (Davis & Perkins, 2002;Dunlap et al, 2007) and PCD can be induced by different means (Castro et al, 2008;Glass & Dementhon, 2006;Lu, 2006). Moreover, the ongoing project for the disruption of all predicted genes in the N. crassa genome (Colot et al, 2006) allows direct testing of the sensitivity of specific mutants to PCDinducing stimuli, including mutants in genes identified by expression profiling.…”

“…Both nuclear and cytoplasmic genes coding for mitochondrial proteins were highly enriched in the gene set repressed by PHS treatment (Supplementary Table S1, page 3). The statistical significance of this enrichment can be inferred from the extremely low P-values (Table 1).Comparison of the wild-type and nuo14 response to H 2 O 2We previously determined that strains containing mutations in complex I were more resistant to treatment with PHS or H 2 O 2 , although strains containing mutations in other components of the respiratory chain did not show a similar resistance to either treatment (Castro et al, 2008). These observations suggest that the resistance of the complex I mutants to PHS and to H 2 O 2 include similar mechanisms.…”