Involvement of cytochrome c release and caspase-3 activation in the oxidative stress-induced apoptosis in human tendon fibroblasts

Abstract

Our previous studies have demonstrated that oxidative stress and apoptosis are involved in human tendon degeneration. The objectives of our current study were to investigate the effect of oxidative stress on human tendon cell apoptosis, and to explore pathways by which tendon cell apoptosis was induced. In vitro oxidative stress was created by exposure of cultured human rotator cuff tendon cells to H(2)O(2). Apoptotic cells were assessed by Annexin V-FITC staining and necrotic cells by propidium iodide (PI) staining using flow cytometry. Cytochrome c and caspase-3 protein expression were detected by Western blotting. A mini-dialysis unit was employed to increase the protein concentration of the cytosolic fraction. Caspase-3 activity was determined by a colorimetric assay. Tendon cell apoptosis induced by H(2)O(2) was both dose and time dependent. Addition of H(2)O(2) resulted in the release of cytochrome c to the cytosol, and an increase of caspase-3 activity and the expression of caspase-3 subunit. The data suggest that oxidative stress-induced apoptosis in human tendon fibroblasts is mediated via pathway(s) that includes release of cytochrome c from mitochondria to the cytosol and activation of caspase-3.