Tetracaine induces apoptosis through a mitochondrion-dependent pathway in human corneal stromal cells in vitro

Abstract

Purpose: Tetracaine is a local anesthetic widely used in ocular diagnosis and ophthalmic surgery and may lead to some adverse effects and complications at a clinical dose. To assess the cytotoxicity and molecular toxicity mechanisms of tetracaine, we used human corneal stromal (HCS) cells as an in vitro model to study the effects of tetracaine on HCS cells.Materials and methods: The cytotoxicity of tetracaine on HCS cells was investigated by examining the changes of cell growth, morphology, viability and cell cycle progressing when HCS cells were treated with tetracaine at concentrations from 10 g/L to 0.078125 g/L. To prove the hypothesis that the cytotoxicity of tetracaine on HCS cells was related with apoptosis induction, we further detected multiple changes in HCS cells, including plasma membrane (PM) permeability, phosphatidylserine (PS) orientation, genomic DNA integrality, and cell ultrastrcuture after treated with tetracaine. Furthermore, the pro-apoptotic signalling pathway induced by tetracaine was explored through detecting the activation of various caspases, the changes of mitochondrial transmembrane potential (MTP), the expression level of Bcl-2 family proteins and the amount of mitochondria-released apoptosis regulating proteins in cytoplasm.Results: Tetracaine at concentrations above 0.15625 g/L had a dose- and time-dependent cytotoxicity to HCS cells, which resulted cell growth inhibition, proliferation retardation, morphological abnormalities and decreased viability. Meanwhile, we found that the HCS cells treated with tetracaine had typical features associated with apoptosis, including an increase in PM permeability, PS externalization, DNA fragmentation and apoptotic body formation. Tetracaine not only resulted in caspase-3, caspase-8 and caspase-9 activation and disruption of MTP but also downregulated Bcl-2 and Bcl-xL and upregulated Bad and Bax, along with the upregulation of cytoplasmic cytochrome c (Cyt. c) and apoptosis-inducing factor (AIF).Conclusions: These results suggested that tetracaine-induced apoptosis might be triggered through Fas death receptors and mediated by Bcl-2 family proteins in the mitochondria-dependent pathway. Our findings identified the cytotoxicity and molecular mechanisms of tetracaine, which could provide a reference value for the safety of this medication and prospective therapeutic interventions in eye clinic.