Inducing effect of hydroxycamptothecin on autophagy of human Tenon capsule fibroblasts in vitro

Authors: Xu Xinyu,  Tong Jun,  Fan Shuxin,  Yuan Zhilan
DOI: 10.3760/cma.j.issn.2095-0160.2015.03.002
Published 2015-03-10
Cite as Chin J Exp Ophthalmol, 2015,33(3): 196-200.

Abstract                              [Download PDF] [Read Full Text]

Background

The fibrosis of filtering area caused by proliferation of human Tenon fibroblasts (HTFs) is one of failure causes following glaucoma surgery.Researches revealed that hydroxycamptothecin can induce the apoptosis of HTFs, but its influence on autophagy of HTFs is unclear.

Objective

This study attempted to investigate whether hydroxycamptothecin can cause an alteration of autophagic activity in HTFs.

Methods

Human Tenon capsular tissue was obtained from 3 patients during strabismus correction surgery under the informed consent of patients and their parents for the primary culture and passaged of HTFs in DMEM containing 10% fetal bovine serum.The generation 3 to 6 cells then were incubated with 0.0, 0.5, 1.0, 4.0, 10.0 mg/L hydroxycamptothecin for 24 hours, respectively.A cell counting kit-8 (CCK-8) was used to detect the cell viability in different treated groups.The autophagic activity of HTFs was evaluated by a Cyto-ID autophagy detection kit, and then the autophagic flux was evaluated by counting the Cyto-ID positive cells under a fluorescence microscope, and the green fluorescence intensity was determined by flow cytometry.Quantitative reverse transcriptase PCR (qRT-PCR) and Western blot analysis were employed to assay the relative expressions of autophagic-associated genes and their proteins in HTFs, including Beclin-1, autophagy related gene 5(ATG-5) and light chain 3(LC-3).

Results

The cell viability of HTFs in the 0.0, 0.5, 1.0, 4.0 and 10.0 mg/L hydroxycamptothecin groups were (100.00±6.44)%, (91.70±6.36)%, (81.47±6.00)%, (68.43±6.69)% and (59.97±6.98)% respectively, showing a gradually declining trend with the increase of hydroxycamptothecin doses, with a significant difference among them (F=19.040, P<0.001), and the viability of HTFs in the 1.0, 4.0 and 10.0 mg/L hydroxycamptothecin groups were significantly decreased than the control group (P<0.05, P<0.01, P<0.01). qRT-PCR analysis revealed that the relative expression levels of Beclin-1 mRNA, ATG-5 mRNA and LC-3 mRNA in 4.0 mg/L hydroxycamptothecin group were (3.225±0.346), (2.839±0.418) and (3.761±0.224) folds higher than those of the control group.The expressions of Beclin-1 and ATG-5 proteins were significantly increased in the 4.0 mg/L hydroxycamptothecin group in comparison with the control group, and the expression intensity ratio of LC-3-Ⅱ/Ⅰwas 0.965±0.159 in the hydroxycamptothecin group, which was significantly higher than 0.275±0.860 of the control group(P=0.003). Cyto-ID staining showed that the percentage of autophagic cells increased dramatically from (11.333±4.010) % to (55.000±9.013) % upon the exposure of HTFs to 4.0 mg/L hydroxycamptothecin (P=0.002). Flow cytometry analysis showed that the green fluorescence intensity in the 4.0 mg/L hydroxycamptothecin group was (3.037±0.513) fold relative to that in the control group, showing a significant difference between the two groups (P=0.003).

Conclusions

Hydroxycamptothecin can induce autophagy in HTFs in vitro.

Key words:

Autophagy; Hydroxycamptothecin; Human, Tenon capsule; Fibroblast; Cell culture; Glaucoma

Contributor Information

Xu Xinyu
Department of Ophthalmology, Affiliated Jiangsu Province Hospital of Nanjing Medical University, Nanjing 210029, China
Tong Jun
Fan Shuxin
Yuan Zhilan
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