To investigate the underlying mechanism by which pirfenidone blocks the transition from the G1 to S phase in primary human Tenon's fibroblasts.
Methods:
Primary human Tenon's fibroblasts were characterized by immunocytofluorescence staining with vimentin, fibroblast surface protein, and cytokeratin. After treating Tenon's fibroblasts with pirfenidone under proliferation conditions (10% fetal bovine serum), cell proliferation was measured using a WST-1 assay. Progression through the cell cycle was analyzed by flow cytometry. The expression of CDK2, CDK6, cyclinD1, cyclinD3, and cyclinE and the phosphorylation of AKT, ERK1/2/MAPK, JNK/MAPK, and p38 MAPK were estimated using western blot analysis.
Results:
Under proliferative conditions, pirfenidone inhibited Tenon's fibroblasts proliferation and arrested the cell cycle at the G1 phase; decreased the phosphorylation of AKT, GSK3β, ERK1/2/MAPK, and JNK/MAPK; increased the phosphorylation of p38 MAPK; and inhibited CDK2, CDK6, cyclin D1, cyclin D3, and cyclin E in a dose-dependent manner. Inhibitors of AKT (LY294002), ERK1/2 (U0126), and JNK (SP600125) arrested the G1/S transition, similar to the effect of pirfenidone. The p38 inhibitor (SB202190) decreased the G1-blocking effect of pirfenidone. The expression of CDK2, CDK6, cyclin D1, and cyclin D3 were inhibited by LY294002, U0126, and SP600125. SB202190 attenuated the pirfenidone-induced reduction of CDK2, CDK6, cyclin D1, cyclin D3, and cyclin E.
Conclusions:
Pirfenidone inhibited HTFs proliferation and induced G1 arrest by downregulating CDKs and cyclins involving the AKT/GSK3β and MAPK signaling pathways.
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References
1.
QuigleyH.A., and BromanA.T.The number of people with glaucoma worldwide in 2010 and 2020. Br. J. Ophthalmol., 90:262–267, 2006.
2.
LichterP.R., MuschD.C., GillespieB.W., et al.Interim clinical outcomes in the Collaborative Initial Glaucoma Treatment Study comparing initial treatment randomized to medications or surgery. Ophthalmology. 108:1943–1953, 2001.
3.
BurrJ., Azuara-BlancoA., AvenellA., and TuulonenA.Medical versus surgical interventions for open angle glaucoma. Cochrane Database Syst. Rev., 9:CD004399, 2012.
4.
AtreidesS.P., SkutaG.L., and ReynoldsA.C.Wound healing modulation in glaucoma filtering surgery. Int. Ophthalmol. Clin., 44:61–106, 2004.
5.
DanielsJ.T., OcclestonN.L., CrowstonJ.G., and KhawP.T.Effects of antimetabolite induced cellular growth arrest on fibroblast-fibroblast interactions. Exp. Eye Res., 69:117–127, 1999.
6.
JampelH.D.Effect of brief exposure to mitomycin C on viability and proliferation of cultured human Tenon's capsule fibroblasts. Ophthalmology. 99:1471–1476, 1992.
7.
KhawP.T., SherwoodM.B., MacKayS.L., RossiM.J., and SchultzG.Five-minute treatments with fluorouracil, floxuridine, and mitomycin have long-term effects on human Tenon's capsule fibroblasts. Arch Ophthalmol. 110:1150–1154, 1992.
8.
AssalianA., ScroggsM.W., ShieldsM.B., ShelburneJ.D., ThrelkeldA.B., and WornialloE.Histology of conjunctival vascular endothelium after filtering surgery with mitomycin C in rabbits. Can. J. Ophthalmol., 31:289–295, 1996.
9.
HeapsR.S., NordlundJ.R., Gonzalez-FernandezF., RedickJ.A., and ConwayB.P.Ultrastructural changes in rabbit ciliary body after extraocular mitomycin C. Invest. Ophthalmol. Vis. Sci., 39:1971–1975, 1998.
10.
JampelH.D., PasqualeL.R., and DibernardoC.Hypotony maculopathy following trabeculectomy with mitomycin C. Arch Ophthalmol. 110:1049–1050, 1992.
11.
WaheedS., RitterbandD.C., GreenfieldD.S., LiebmannJ.M., SidotiP.A., and RitchR.Bleb-related ocular infection in children after trabeculectomy with mitomycin C. Ophthalmology. 104:2117–2120, 1997.
12.
Palanca-CapistranoA.M., HallJ., CantorL.B., MorganL., HoopJ., and WuDunnD.Long-term outcomes of intraoperative 5-fluorouracil versus intraoperative mitomycin C in primary trabeculectomy surgery. Ophthalmology. 116:185–190, 2009.
13.
SpagnoloP., SverzellatiN., RossiG., et al.Idiopathic pulmonary fibrosis: an update. Ann. Med., 47:15–27, 2015.
14.
LedererD.J., BradfordW.Z., FaganE.A., et al.Sensitivity analyses of the change in FVC in a Phase 3 trial of pirfenidone for idiopathic pulmonary fibrosis. Chest. 148:196–201, 2015.
15.
Castro-TorresR.D., Chaparro-HuertaV., Flores-SotoM.E., et al.Pirfenidone attenuates microglial reactivity and reduces inducible nitric oxide synthase mRNA expression after kainic acid-mediated excitotoxicity in pubescent rat hippocampus. J. Mol. Neurosci., 56:245–254, 2015.
16.
WestraI.M., OosterhuisD., GroothuisG.M., and OlingaP.The effect of antifibrotic drugs in rat precision-cut fibrotic liver slices. PLoS One. 9:e95462, 2014.
17.
TampeD., and ZeisbergM.Potential approaches to reverse or repair renal fibrosis. Nat. Rev. Nephrol., 10:226–237, 2014.
18.
YamagamiK., OkaT., WangQ., et al.Pirfenidone exhibits cardioprotective effects by regulating myocardial fibrosis and vascular permeability in pressure-overloaded hearts. Am. J. Physiol. Heart Circ. Physiol., 309:H512–522, 2015.
19.
ZhongH., SunG., LinX., WuK., and YuM.Evaluation of pirfenidone as a new postoperative antiscarring agent in experimental glaucoma surgery. Invest. Ophthalmol. Vis. Sci., 52:3136–3142, 2011.
20.
SunG., LinX., ZhongH., et al.Pharmacokinetics of pirfenidone after topical administration in rabbit eye. Mol. Vis., 17:2191–2196, 2011.
21.
LinX., YuM., WuK., YuanH., and ZhongH.Effects of pirfenidone on proliferation, migration, and collagen contraction of human Tenon's fibroblasts in vitro. Invest. Ophthalmol. Vis. Sci., 50:3763–3770, 2009.
22.
MalumbresM., and BarbacidM.Cell cycle, CDKs and cancer: a changing paradigm. Nat. Rev. Cancer., 9:153–166, 2009.
23.
LimS., and KaldisP.Cdks, cyclins and CKIs: roles beyond cell cycle regulation. Development. 140:3079–3093, 2013.
24.
HeZ., DengY., LiW., et al.Overexpression of PTEN suppresses lipopolysaccharide-induced lung fibroblast proliferation, differentiation and collagen secretion through inhibition of the PI3-K-Akt-GSK3beta pathway. Cell Biosci. 4:2, 2014.
25.
HeX.Q., ChenR., YangP., LiA.P., ZhouJ.W., and LiuQ.Z.Biphasic effect of arsenite on cell proliferation and apoptosis is associated with the activation of JNK and ERK1/2 in human embryo lung fibroblast cells. Toxicol. Appl. Pharmacol., 220:18–24, 2007.
26.
ToddD.E., DenshamR.M., MoltonS.A., et al.ERK1/2 and p38 cooperate to induce a p21CIP1-dependent G1 cell cycle arrest. Oncogene. 23:3284–3295, 2004.
27.
LiaoK., LiJ., and WangZ.Dihydroartemisinin inhibits cell proliferation via AKT/GSK3beta/cyclinD1 pathway and induces apoptosis in A549 lung cancer cells. Int. J. Clin. Exp. Pathol., 7:8684–8691, 2014.
28.
ConteE., GiliE., FagoneE., FrucianoM., IemmoloM., and VancheriC.Effect of pirfenidone on proliferation, TGF-beta-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts. Eur. J. Pharm. Sci., 58:13–19, 2014.
29.
ChungS.A., JeonB.K., ChoiY.H., BackK.O., LeeJ.B., and KookK.H.Pirfenidone attenuates the IL-1beta-induced hyaluronic acid increase in orbital fibroblasts from patients with thyroid-associated ophthalmopathy. Invest. Ophthalmol. Vis. Sci., 55:2276–2283, 2014.
30.
PengY., YangH., ZhuT., et al.The antihepatic fibrotic effects of fluorofenidone via MAPK signalling pathways. Eur. J. Clin. Invest., 43:358–368, 2013.
31.
FengY.M., FengC.W., ChenS.Y., HsiehH.Y., ChenY.H., and HsuC.D.Cyproheptadine, an antihistaminic drug, inhibits proliferation of hepatocellular carcinoma cells by blocking cell cycle progression through the activation of P38 MAP kinase. BMC Cancer. 15:134, 2015.
32.
CalvoN., MartinM.J., de BolandA.R., and GentiliC.Involvement of ERK1/2, p38 MAPK, and PI3K/Akt signaling pathways in the regulation of cell cycle progression by PTHrP in colon adenocarcinoma cells. Biochem. Cell Biol., 92:305–315, 2014.
33.
AmbrosinoC., and NebredaA.R.Cell cycle regulation by p38 MAP kinases. Biol. Cell., 93:47–51, 2001.
34.
SherrC.J., and RobertsJ.M.CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13:1501–1512, 1999.
35.
LorenziniA., TresiniM., Mawal-DewanM., et al.Role of the Raf/MEK/ERK and the PI3K/Akt(PKB) pathways in fibroblast senescence. Exp. Gerontol., 37:1149–1156, 2002.
36.
ZhangD., and JacobbergerJ.W.TGF-beta 1 perturbation of the fibroblast cell cycle during exponential growth: switching between negative and positive regulation. Cell Prolif. 29:289–307, 1996.
37.
KhawP., GrehnF., HolloG., et al.A phase III study of subconjunctival human anti-transforming growth factor beta(2) monoclonal antibody (CAT-152) to prevent scarring after first-time trabeculectomy. Ophthalmology. 114:1822–1830, 2007.
38.
FischerC.V., MansV., HornM., NaxerS., KlettnerA., and van OterendorpC.The Antiproliferative Effect of Bevacizumab on Human Tenon Fibroblasts Is Not Mediated by Vascular Endothelial Growth Factor Inhibition. Invest. Ophthalmol. Vis. Sci., 57:4970–4977, 2016.
