Abstract
Purpose: :
This study was designed to investigate whether autophagic process is implicated in apoptotic death of human tenon’s capsule fibroblasts (HTCFs) by mitomycin-C.
Methods: :
Autophagic phenotype is tested by fluorescence microscopy and flow cytometry with specific biological staining dyes including monodansylcadaverine and acridine orange and western blot with microtubule-associated protein 1 light chain 3 (LC3).
Results: :
Treatment with mitomycin-C (0.4mg/mL) increased the apoptosis of HTCFs, which was characterized as fragmentation of nucleic acid and genomic DNA, chromatin condensation, and increase in sub-G(0)/G(1) fraction of cell cycle. The author also found that both autophagy phenotype and reactive oxygen species(ROS) generation were observed in mitomycin-C treated HTCFs. Pharmacologic inhibition of autophagy suppressed the HTCFs ability to ROS generation. However, scavenging of ROS with NAC and and GSH did not abolish the formation of acidic vesicle organelle in mitomycin-C treated HTCFs. Moreover, calcium release with other evidence of endoplasmic reticulum(ER) stress occurred by mitomycin-C induced autophagic events which are preceded by ROS generation and ER stress in HTCFs.
Conclusions: :
Apoptosis of HTCFs by mitomycin-C was mediated by early autophagy formation accompanying ROS generation and ER stress activation
Keywords: apoptosis/cell death • cell survival • wound healing