Effects Of Genistein On Induction Apoptosis In Cultured Retinal Pigment Epithelial Cells

Retinal pigment epithelial (RPE) cells account for a number ofvital functions required for the maintenance of retinal integrity,such as the adhesion of the retina. A lack of adhesion between RPEcells and the Bruch's membrane has well-characterized pathologicconsequences. For example, a breakdown in adhesion between thephotoreceptor cell layer of the neural retina and the RPE eventuallyleads to photoreceptor degeneration and blindness. A lack ofadhesion between the RPE and the choroid results in proliferativevitreoretinopathy (PVR). During wound healing in diabeticretinopathy, eye injury, inflammatory eye disease, untreated or surgically treated retinal detachment, PVR is an undesirablecomplication. In PVR, RPE cells act in a wound healing fashion.Indeed, these normally quiescent and differentiated cellsdetach from the underlying Bruch's membrane, the earlypathogenesis involves proliferation of RPE cells. Thus theinhibition of RPE cells proliferation is likely to be the possiblefuture treatment of the proliferative retinal diseases, such as PVR.Genistein, an isoflavone that is plentiful in soy beans, on theinhibition of cell proliferation is induced by various mechanisms. Itblocks the cell cycle at the G1/S transition, arrests the cell cycle inG2/M phase and induces apoptosis in various cells. Genisteinhas also been reported to has an attractive safety profile, with anIC50 for endothelial cells of 12.5μM and an LC50 of＞300μM.It has been conducted numerous investigations of genistein inanimal models of retinal disease, in which is an effective compoundfor long-term oral dosing and is effective in ameliorating thebiochemical, histopathologic, and morphologic changes in experimental retinal disease states.To investigate whethergenistein has a therapeutic effect on PVR, we attempted to treatRPE cells with genistein in vitro. As part of this study we examinedalso the mechanisms by which genistein is taken up by RPE cells.Objective:Human RPE cells play an important role in retinal physiologyand pathology. These cells migrate and proliferate, lead to abreakdown in adhesion between the RPE and the neural retinaresults in proliferative vitreoretinopathy (PVR). To investigate theapoptosis induction effect of genistein on cultured human RPE cellsin vitro.Methods:To investigate whether genistein has a therapeutic effect onPVR, we attempted to treat a human RPE cell line (designated ARPE-19) with genistein in vitro. After plating, RPE cells wereincubated with various concentrations of genistein (0,25,50,100and 200umol/L) for 24h. The morphologic changes characteristicof apoptosis were observed under light microscopy. The cellproliferation was determined by MTT assay. To investigate themechanisms involved in growth inhibition induced by genistein,RPE cells were subjected to cell cycle analysis byfluorescence-activated cell analyzer (FACS) and a cytofluorimetricanalysis using PI and Annexin V-FITC double staining to confirmthe induction of apoptosis. Since Mitogen-activated protein kinase(MAPK) signaling pathway was one of the most significantlyaffected by genistein treatment, the activation of MAPK wasmeasured by western blot.Results:The normal RPE cells appear flat and multi-angular, with a fewpigments dwelling in the cytoplasm. 24 hours after adding genistein in different concentrations, compared with normal(1)the cells shrinked, cytoplasm condensed and density increasedcompared with controls. Some of the cells will detach and float inthe media(2)Cell growth was inhibited compared with normal. Genistein indifferent concentrations displayed an anti-proliferation capability onRPE cells with growth inhibitory rate of 6.44±2.0,14.71±0.8,28.97±1.5,45.75±0.9. The effects are highly dose-dependent.(3)Treatment with genistein significantly reduced proliferation of RPEcell with a progressive increased population in G2/M phase,decreased in G1 phase, accompanied by an increase in S andapoptotic population, in a dose-dependent manner. Our dataindicated that genistein involved in apoptosis induction effect onRPE cells is owing to the G2/M arrested.(4) 24 hours after genistein treatment, the apoptotic rate of(16.87±1.3)％,(19.44±0.9)％,(23.14±2.4)％,(34.81±2.8)％. Theapoptotic rate is highly genistein concentration dependent. (5)The activation of ERK1/2 and p38MAPK were impaired. Our dataindicated that genistein involved in the apoptosis induction effect onRPE cells is owing to the inactivation of ERK1/2, and maybe owingto the inactivation of p38. No changes of JNK and BMK1 expressionand phosphorylation were detected in all groups, indicating theabsence of JUK and BMK1 in genistein induced RPE cell apoptosis.Conclusion:Genistein in different concentrations has a dose-dependentanti-proliferation and apoptosis induction effect on RPE cells thatmay relate with ERK signaling pathway. Genistein may prove usefulas an inhibitor of RPE cells undergoing progressive PVR.