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Abstract
Purpose
To evaluate the anti-fibrotic effects of nilotinib on the survival of cultured human Tenon's capsule fibroblasts (HTFs).
Methods
HTF primary cultures were obtained from samples following glaucoma surgery. Primarily cultured HTFs were exposed to 1, 5, 10, and 20 µM nilotinib for 24 hours. The effects of nilotinib on HTF proliferation and cell viability were determined using the 3-(4,5-dimethylthiazone-2-yl)-2,5-diphenyl tetrazolium (MTT) assay, and apoptosis was determined by flow cytometry using annexin-V/propidium iodide (PI) double staining. Apoptosis-related proteins were detected by western blotting.
Results
The MTT assay showed that nilotinib induced an inhibition of HTF proliferation at concentrations of 10 and 20 µM (p < 0.001 and p < 0.001, respectively). Annexin V/PI double staining showed significantly increased apoptosis in cells treated with nilotinib. Nilotinib activated caspase-3, -9, and poly adenosine diphosphate ribose polymerase cleavage, and downregulated the expression of B-cell lymphoma-extra large and Bax, which indicated that nilotinib-induced apoptosis was partly mediated through the mitochondrial pathway. In addition, treatment with nilotinib decreased the expression of α-smooth muscle actin and transforming growth factor-β.
Figures and Tables
Figure 1
MTT assay of nilotinib-treated human tenon's capsule fibroblasts. Nilotinib decreased cellular viability significantly in a dose-dependent manner. The results represent the means of six independent experiments. MTT = 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide. *p<0.005 vs. control; **p<0.001 vs. control (Kruskal-Wallis test with post hoc pairwise comparisons adjusted by the Bonferroni method).
Figure 2
Apoptosis assay of human Tenon's capsule fibroblasts by flow cytometry. Annexin V/propidium iodide (PI) dual-staining demonstrated significantly increased apoptosis (12.4% [A], 31.5% [B], and 69.4% [C], respectively) in cells treated with nilotinib for 24 hours. Results were confirmed in at least two independent experiments.
Figure 3
Expression levels of caspase-3, caspase-9, poly ADP ribose polymerase with (24 hours) or without nilotinib determined by Western blot analysis. Western blots (A) and relative band intensity values (B) show that nilotinib induces caspase activation in human Tenon's capsule fibroblasts. β-actin was selected as the loading control. The results represent the means of three independent experiments. ADP = adenosine diphosphate; PARP = poly ADP ribose polymerase. *p<0.005 vs. control; **p<0.001 vs. control (Kruskal-Wallis test with post hoc pairwise comparisons adjusted by the Bonferroni method).
Figure 4
Expression levels of Bcl-xL, Bax, α-smooth muscle actin (α-SMA), TGF-β with (24 hours) or without nilotinib determined by Western blot analysis. (A) Nilotinib treatment resulted in a downregulation of Bcl-xL, Bax. (B) Nilotinib significantly downregulated expression of α-SMA, TGF-β. β-actin was selected as the loading control. The results represent the means of three independent experiments. Bcl-xL = B-cell lymphoma-extra large; TGF-β = Transforming growth factor beta. *p<0.005 vs. control; **p<0.001 vs. control (Kruskal-Wallis test with post hoc pairwise comparisons adjusted by the Bonferroni method).
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