Kaempferol induces programmed cell death in Naegleria fowleri

Background: Naegleria fowleri is a brain-eating amoeba causing a fatal brain infection called prima r y amoebic meningoencephalitis (PAM). Despite its high mortality over 95%, effective therapeutic drug for PAM has not been developed yet. Therefore, development of an effective and safe therapeutic drug for PAM is urgently needed. In this study, we investigated anti-amoebic effect of kaempferol (KPF) against N. fowleri and its underlying anti-amoebic molecular mechanisms. Methods: Anti-amoebic activity of KPF against N. fowleri trophozoites, as wel l as cytotoxicity of KPF in C6 glial cells and CHO-K1 cells were investigated. The programmed cell death mechanisms in KPF-treated N. fowleri were also analyzed by apoptosis-necrosis assay, mitochondrial dysfunction assay, TUNEL assay, RT-qPCR , and CYTO-ID assay. Results: KPF showed anti-amoebic activity against N. fowleri trophozoites with an IC50 of 29.28 & PLUSMN; 0.63 mu M. However, it showed no significant cytotoxicity to mammalian cells. KPF induced significant morphological al-terations of the amoebae, resulting in death. Signals associated with apoptosis were detected in the amoebae upon treatment with KPF. KPF induced an increase of intracellular reactive oxygen species level , loss of mito-chondrial membrane potential , increases of expression levels of genes associated with mitochondria dysfunction, and reduction of ATP levels in the amoebae. Autophagic vacuole accumulations with increased expression levels of autophagy-related genes were also detected in KPF-treated amoebae. Conclusion: KPF induces programmed cell death in N. fowleri trophozoites via apoptosis-like pathway and autophagy pathway. KPF cou l d be used as a candidate of anti-amoebic drug or supplement compound in the process of developing or optimizing therapeutic drug for PAM.