Hydroxycamptothecin induces apoptosis of human tenon's capsule fibroblasts by activating the PERK signaling pathway

Xue Yin, Hong Sun, Dongyi Yu, Ya Liang, Zhilan Yuan, Yingbin Ge
Investigative Ophthalmology & Visual Science 2013 July 16, 54 (7): 4749-58

PURPOSE: Hydroxycamptothecin (HCPT) has been proven to induce apoptosis in fibroblasts. In this study, we investigated whether the PRKR-like ER kinase (PERK) pathway is implicated in apoptotic signaling of human Tenon's capsule fibroblasts (HTCFs) by HCPT.

METHODS: Normal and PERK-knockdown HTCFs were used in this study. Apoptosis was determined by the cell viability assay, Annexin V/propidium iodide (PI) dual-staining, cell cycle analysis in HTCFs treated with HCPT in various doses and for various durations. Endoplasmic reticulum (ER) stress markers and sensor proteins were detected by Western blot analysis. Mitochondrial dysfunction was measured by detecting the mitochondrial membrane potential (ΔΨm) and measuring the expression of cytochrome c (cyt c).

RESULTS: HCPT induced apoptosis in the HTCFs, which was characterized as decreased cell viability and sub-S fraction of the cell cycle and increased apoptosis rate by Annexin V/PI dual-staining. The activity levels of caspase-3 and caspase-9 were significantly increased and were accompanied by cytosolic release of cyt c and decreased ΔΨm in response to HCPT. Treatment with HCPT increased the expression of glucose-regulated protein 78 (GRP78), phospho-PERK, activating transcription factor 6 (ATF6), phosphoinositol-requiring kinase 1 (IRE1), C/EBP homologous protein (CHOP), Bax, and phospho-c-Jun N-terminal kinase (JNK) and decreased the expression of Bcl-2. Knockdown of PERK attenuates HCPT-induced apoptosis in HTCFs, dependent upon both ER stress and the mitochondrial apoptotic pathway.

CONCLUSIONS: This study suggests that the ER stress response and mitochondrial dysfunction are involved in apoptosis induced by HCPT in HTCFs, which might be mediated by PERK; thus, this study offers new insight into preventing postoperative scarring via treatment with HCPT.

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