Cytotoxic Effect of Latanoprost on Human Corneal Stromal Cells in vitro and its Possible Mechanisms

ABSTRACT

Purpose: To investigate the cytotoxic effect of latanoprost on corneal stroma and its underlying cellular and molecular mechanisms using non-transfected human corneal stromal (HCS) cells as an in vitro model.

Methods: After HCS cells were treated with latanoprost at concentrations varying from 50 mg/l (clinical therapeutic dosage) to 0.78125 mg/l, and cell morphology, cell viability, and cell cycle were detected by light microscopy, methyl thiazolyl tetrazolium assay, and flow cytometry (FCM) with propidium iodide (PI) staining, respectively. Meanwhile, alterations in plasma membrane permeability, phosphatidylserine (PS) orientation, DNA integrality, and cell ultrastructure were examined by acridine orange (AO)/ethidium bromide (EB) double staining, FCM with Annexin-V/propidium iodide (PI) staining, DNA electrophoresis, and transmission electron microscopy. Furthermore, caspase activation, mitochondrial transmembrane potential (MTP), and expression of pro-apoptotic regulators were determined by ELISA, FCM with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethybenzimida (JC-1) staining, and Western blot, respectively.

Results: Latanoprost above concentrations of 3.125 mg/l can induce dose- and time-dependent morphological abnormality, growth retardation, viability decline, and plasma membrane permeability elevation of HCS cells. Moreover, latanoprost can arrest the cell cycle of these cells at S phase and induce PS externalization, DNA fragmentation, and apoptotic body formation of the cells. Furthermore, latanoprost can induce activation of caspase-3, -8 and -9; disruption of MTP; downregulation of anti-apoptotic Bcl-2; upregulation of pro-apoptotic Bax; and cytoplasmic cytochrome c release.

Conclusions: Latanoprost above and at 3.125 mg/l (1/16 of its clinical therapeutic dosage) has a dose- and time-dependent cytotoxicity to HCS cells by inducing death receptor-mediated mitochondria-dependent apoptosis, which should be used with great caution in clinical situations to avoid undesired damages to HCS cells.

KEYWORDS:

• Apoptosis
• cytotoxicity
• human corneal stromal cells
• latanoprost
• mitochondrion-dependent pathway

Acknowledgment

We want to thank Prof. Ming-Zhuang Zhu (Key Laboratory of Mariculture, Fisheries College, Ocean University of China) for his excellent contribution to flow cytometry analysis of this study.

Funding

This work was supported by the National High Technology Research and Development Program (“863” Program) of China (No. 2006AA02A132) and China Postdoctoral Science Foundation (2013M541960).

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Supplemental Material

Supplementary data, including FCM images of PI staining photographs and Annexin-V/PI staining photographs of latanoprost-exposed HCS cells, is available on the publisher's website.

Additional information

Funding

This work was supported by the National High Technology Research and Development Program (“863” Program) of China (No. 2006AA02A132) and China Postdoctoral Science Foundation (2013M541960).