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

ABSTRACT: 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.

SUBMITTER: Fan WY

PROVIDER: S-EPMC6062160 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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

Fan Wen Yi WY Sui Yun Long YL Fan Ting Jun TJ

Toxicology research 20160712 5

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 molecu ...[more]

PMID: 30090447

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Project description:Extensive research has been done in the search for innovative treatments against colon adenocarcinomas; however, the incidence rate of patients remains a major cause of cancer-related deaths in Malaysia. Natural bioactive compounds such as curcumin have been substantially studied as an alternative to anticancer drug therapies and have been surmised as a potent agent but, nevertheless, remain deficient due to its poor cellular uptake. Therefore, efforts now have shifted toward mimicking curcumin to synthesize novel compounds sharing similar effects. A synthetic analog, (Z)-3-hydroxy-1-(2-hydroxyphenyl)-3-phenylprop-2-ene-1-one (DK1), was recently synthesized and reported to confer improved bioavailability and selectivity toward human breast cancer cells. This study, therefore, aims to assess the anticancer mechanism of DK1 in relation to the induction of in vitro cell death in selected human colon cancer cell lines. Using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, the cytotoxicity of DK1 towards HT29 and SW620 cell lines were investigated. Acridine orange/propidium iodide (AO/PI) dual-staining assay and flow cytometry analyses (cell cycle analysis, Annexin/V-FITC and JC-1 assays) were incorporated to determine the mode of cell death. To further determine the mechanism of cell death, quantitative real-time polymerase chain reaction (qRT-PCR) and proteome profiling were conducted. Results from this study suggest that DK1 induced changes in cell morphology, leading to a decrease in cell viability and subsequent induction of apoptosis. DK1 treatment inhibited cell viability and proliferation 48 h post treatment with IC50 values of 7.5 ± 1.6 µM for HT29 cells and 14.5 ± 4.3 µM for SW620 cells, causing cell cycle arrest with increased accumulation of cell populations at the sub-G₀/G₁phaseof 74% and 23%, respectively. Flow cytometry analyses showed that DK1 treatment in cancer cells induced apoptosis, as indicated by DNA fragmentation and depolarization of the mitochondrial membrane. qRT-PCR results show significant upregulation in the expression of caspase-9 in both HT29 and SW620 cell lines, further supporting that cell death induction by DK1 is via an intrinsic pathway. These outcomes, therefore, demonstrate DK1 as a potential anticancer agent for colon adenocarcinoma due to its anti-apoptotic attributes.

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

Publications

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

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