Gatifloxacin inducing apoptosis of stromal fibroblasts through cross-talk between caspase-dependent extrinsic and intrinsic pathways.

AIM: To reveal the cytotoxicity and related mechanisms of gatifloxacin (GFX) to stromal fibroblasts (SFs) in vitro. METHODS: SFs were treated with GFX at different concentrations (0.009375%-0.3%), and their viability was detected by MTT method. The cell morphology was observed using light/transmission electron microscope. The plasma membrane permeability was measured by AO/EB double-staining. Then cell cycle, phosphatidylserine (PS) externalization, and mitochondrial transmembrane potential (MTP) were analyzed by flow cytometry. DNA damage was analyzed by electrophoresis and immunostaining. ELISA was used to evaluate the caspase-3/-8/-9 activation. Finally, Western blotting was applied for detecting the expressions of apoptosis-related proteins. RESULTS: Morphological changes and reduced viability of GFX-treated SFs demonstrated that GFX above 0.009375% had cytotoxicity to SFs with dependence of concentration and time. GFX-treating cells also showed G1 phase arrest, increased membrane permeability, PS externalization and DNA damage, which indicated that GFX induced apoptosis of SFs. Additionally, GFX could activate the caspase-8, caspase-9, and caspase-3, induce MTP disruption, downregulate B-cell leukemia-2 (Bcl-2) and B-cell leukemia-XL (Bcl-XL), and upregulate Bcl-2 assaciated X protein (Bax), Bcl-2-associated death promoter (Bad), Bcl-2 interacting domain (Bid) and cytoplasmic cytochrome C in SFs, suggesting that caspase-dependent extrinsic and intrinsic pathways were related to GFX-contributed apoptosis of SFs. CONCLUSION: The cytotoxicity of GFX induces apoptosis of SFs through triggering the caspase-dependent extrinsic and intrinsic pathways.

Proparacaine induces cytotoxicity and mitochondria-dependent apoptosis in corneal stromal cells both in vitro and in vivo.

Proparacaine (PPC) is a widely used topical anaesthetic in the eye clinic; its abuse may damage the cornea and result in impairment of vision. Although PPC has been reported to be cytotoxic to human keratocytes, there is no scientific report about its toxic mechanisms in human corneal stroma. Here, we evaluated the cytotoxicity of PPC to corneal stroma in an in vitro model of human corneal stromal (HCS) cells and an in vivo model of cat corneas. To postulate the cellular and molecular mechanisms involved in PPC toxicity, changes in the hallmarks of apoptosis as well as in pro-apoptotic signaling pathways were investigated. Our results showed that PPC at concentrations varying from 5.0 to 0.15625 g L-1 induced dose- and time-dependent cell atrophy, vacuolation, cytopathic effects, and viability decline in vitro. Moreover, PPC induced G1 phase arrest, plasma membrane permeability elevation, phosphatidylserine externalization, DNA fragmentation, chromatin condensation, and apoptotic body formation of HCS cells. Furthermore, PPC could induce caspase-2, -3 and -9 activation, and mitochondrial transmembrane potential disruption. Expression of Bcl-xL and Bax were downregulated and upregulated, respectively, and cytoplasmic cytochrome c and apoptosis inducing factor were upregulated remarkably after PPC treatment. The cytotoxicity and pro-apoptotic effects of PPC were also proven by induced corneal edema, apoptotic-like ultrastructural alterations and DNA fragmentation of keratocytes in cat corneas in vivo. These results suggest that PPC above 1/32 of its clinical dosage has remarkable cytotoxicity to corneal stromal cells, which is achieved by inducing death receptor-mediated mitochondria-dependent apoptosis of HCS cells.