OBJECTIVES: The present study was designed to determine whether or not the caspase-independent apoptotic pathway participated in the cellular death of spiral ganglion neurons (SGNs) after exposure to peroxynitrite (ONOO(-)), with particular attention given to the intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) and endonuclease G (Endo G) in this process. METHODS: The rat SGNs were isolated and primary cultured in vitro and were exposed to ONOO(-) with pre-treatment of pan-caspase inhibitor. Morphological changes of SGNs were observed by acridine orange cytochemistry staining, and apoptosis was examined by flow cytometry. The translocation of mitochondrial AIF and Endo G was detected by immunocytochemistry and Western blot. The protein expressions of Bcl-2 family in SGNs exposed to ONOO(-) were determined by Western blot. RESULTS: Treatment of SGNs with ONOO(-) resulted in the occurrence of caspase-independent apoptosis as evidenced by acridine orange staining and flow cytometry analysis. The immunocytochemical analysis showed that AIF and Endo G labeling were marked in neuronal nuclei, while the Western blot demonstrated the intranuclear localization of AIF and Endo G in SGNs treated with ONOO(-). Western blot analysis demonstrated that ONOO(-) increased the Bax expression while reducing Bcl-2 expression, which was not prevented by pre-treatment with caspase inhibitor. CONCLUSION: These data indicate that ONOO(-) can trigger caspase-independent apoptosis in SGNs associated with mitochondrial AIF and Endo G intranuclear localization.
OBJECTIVES: The present study was designed to determine whether or not the caspase-independent apoptotic pathway participated in the cellular death of spiral ganglion neurons (SGNs) after exposure to peroxynitrite (ONOO(-)), with particular attention given to the intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) and endonuclease G (Endo G) in this process. METHODS: The rat SGNs were isolated and primary cultured in vitro and were exposed to ONOO(-) with pre-treatment of pan-caspase inhibitor. Morphological changes of SGNs were observed by acridine orange cytochemistry staining, and apoptosis was examined by flow cytometry. The translocation of mitochondrial AIF and Endo G was detected by immunocytochemistry and Western blot. The protein expressions of Bcl-2 family in SGNs exposed to ONOO(-) were determined by Western blot. RESULTS: Treatment of SGNs with ONOO(-) resulted in the occurrence of caspase-independent apoptosis as evidenced by acridine orange staining and flow cytometry analysis. The immunocytochemical analysis showed that AIF and Endo G labeling were marked in neuronal nuclei, while the Western blot demonstrated the intranuclear localization of AIF and Endo G in SGNs treated with ONOO(-). Western blot analysis demonstrated that ONOO(-) increased the Bax expression while reducing Bcl-2 expression, which was not prevented by pre-treatment with caspase inhibitor. CONCLUSION: These data indicate that ONOO(-) can trigger caspase-independent apoptosis in SGNs associated with mitochondrial AIF and Endo G intranuclear localization.
Authors: Christian Vélez; Jessica Soto; Karoline Ríos; Luz Silva; Wigberto Hernandez; Luis A Rivera; Ana I Ortiz-Colón; Osvaldo Cox; Beatriz Zayas Journal: Open Med Chem J Date: 2017-06-30