Research Article: Silibinin Inhibits Platelet-Derived Growth Factor-Driven Cell Proliferation via Downregulation of N-Glycosylation in Human Tenon’s Fibroblasts in a Proteasome-Dependent Manner

Date Published: December 28, 2016

Publisher: Public Library of Science

Author(s): Yi-Hao Chen, Ching-Long Chen, Da-Wen Lu, Chang-Min Liang, Ming-Cheng Tai, Jiann-Torng Chen, Eve-Isabelle PECHEUR.


The objective of this study was to evaluate the effects of silibinin on cell proliferation in platelet-derived growth factor (PDGF)-treated human Tenon’s fibroblasts (HTFs). The effect of silibinin on cell proliferation in PDGF-treated HTFs was determined by examining the expression of proliferating cell nuclear antigen (PCNA) and performing WST-1 assays. Cell cycle progression was evaluated using flow cytometry. The related cyclins and cyclin-dependent kinases (CDKs) were also analyzed using western blot. A modified rat trabeculectomy model was established to evaluate the effect of silibinin on cell proliferation in vivo. Western blot analysis was carried out to determine the effect of silibinin on the expression of PDGF receptor and on the downstream signaling pathways regulated by PDGF receptor. PDGF elevated the expression of PCNA in HTFs, and this elevation was inhibited by silibinin. The inhibitory effect of silibinin on cell proliferation was also confirmed via WST-1 assay. PDGF-stimulated cell cycle in HTFs was delayed by silibinin, and the related cyclin D1 and CDK4 were also suppressed by silibinin. In the rat model of trabeculectomy, silibinin reduced the expression of PCNA at the site of blebs in vivo. The effects of silibinin on PDGF-stimulated HTFs were mediated via the downregulation of PDGF receptor-regulated signaling pathways, such as ERKs and STATs, which may be partially caused by the downregulation of N-glycosylation of PDGF receptor beta (PDGFRβ). The effect of silibinin on modulation of N-glycosylation of PDGFRβ was mediated in a proteasome-dependent manner. Silibinin inhibited cell proliferation and delayed cell cycle progression in PDGF-treated HTFs in vitro. PDGF also modulated the process of N-glycosylation of the PDGFRβ in a proteasome-dependent manner. Our findings suggest that silibinin has potential therapeutic applications in glaucoma filtering surgery.

Partial Text

Glaucoma is a major cause of irreversible blindness worldwide and presents as a progressive optic atrophy [1–3]. Until date, lowering intraocular pressure (IOP) has been the only successful therapeutic strategy for treating glaucoma. The most common surgical treatment for glaucoma is the procedure known as glaucoma filtering surgery [4]. Glaucoma filtering surgery is performed to create an artificial route to drain the aqueous humor from the anterior chamber to the subconjunctival space, leaving a bleb that is formed in the subconjunctival space [5]. The bleb formation is similar to the wound healing process of soft tissues, which involves inflammation, proliferation, and wound remodeling [6, 7]. Scar formation in the bleb, which is a manifestation of extensive wound healing process, is the major cause of failure in this surgery [8]. To attenuate scar formation, the inhibition of inflammation, proliferation, or wound remodeling has been proposed as a strategy. Several agents have been studied for adjunctive use [9, 10], but none has produced satisfactory results.

In this study, we tested our hypothesis that silibinin inhibits PDGF-induced HTF proliferation in vitro and in vivo and to explore the underlying mechanisms. The results of the present study showed that treatment with silibinin at concentrations of 50 and 100 μM significantly suppressed the PDGF-induced proliferation of HTFs in a dose-dependent manner and delayed the cell cycle progression of HTFs through the G1-S transition without affecting cell viability. The antiproliferative effect of silibinin was also confirmed in a modified animal trabeculectomy model. We provided evidence that silibinin modulated the level of N-glycosylation of PDGFRβ and reduced the downstream signaling pathways, ERK and STATs. Finally, we showed that silibinin promoted the formation of hypo-glycosylated PDGFRβ in a proteasome-dependent manner.

We showed that silibinin inhibits the PDGF-mediated cell proliferation and delays the cell cycle progression in vitro. We present mechanistic evidence that these effects of silibinin result from the downregulation of the PDGFR-regulated signaling, which may, at least partially, be caused by the increased expression of non-functional hypoglycosylated PDGFRβ. We also showed that the 26S proteasomal pathway was involved in this process. Silibinin also abrogated the cell proliferation at the subconjunctival space in the in vivo rat model of trabeculectomy. Our findings provide evidence that silibinin could be a therapeutic candidate as an adjunctive agent to decrease the failure rate of glaucoma filtering surgeries.