Inhibition of anomalous retinal pigment epithelial cell activities, anin vitro study for the effects of 5-fluorouracil and Agaricus bisporuslectin

Abstract

 Proliferative vitreoretinopathy (PVR) remains the major cause of failure of retinal detachment surgery. Retinal pigment epithelial (RPE) cells have been suggested to play a major role in the pathogenesis of PVR. Numerous studies have employed pharmacological means to modulate cellular activities in attempts to inhibit the process. Recent attempts using adjunctive therapy during PVR surgery that consisted of 5-fluorouracil (5-FU) and low molecular weight heparin showed some promise in preventing PVR but the concern is that prolonged 5-FU treatment may have a toxic effect. On the other hand, lectin from the edible mushroom Agaricus bisporus (ABL) was found to inhibit growth of RPE cells in a potent manner without apparent cytotoxicity. This lectin could be a candidate to modulate anomalous proliferation of RPE cells while the mechanism for the observed inhibition is unknown.

In our study, we investigated whether RPE cells treated with 5-FU or ABL would attenuate cellular proliferation, cell migration, cell adhesion and cell-mediated contraction rates. Further, we investigated if complementary inhibition for the above cellular activities could be obtained when RPE cells were treated with ABL after the short treatment using 5-FU. We also explored the possible mechanisms through which ABL inhibited RPE cell proliferation.

ARPE-19 and primary human RPE cells were treated with 5-FU or vehicle for 10 minutes. Cells were then maintained in culture medium supplemented with or without ABL. The rate of cellular proliferation was measured by a tetrazolium salt assay. Effects on cell adhesion were investigated through loading RPE cells onto the strips coated with collagen I or fibronectin. Cell migration was investigated using a scratch wound model. The effect on cell-mediated contraction was assessed using a free floating collagen I matrix. Cytotoxicity of 5-FU and ABL was determined by the live/dead assay.

To elucidate the mechanism through which ABL inhibited RPE cell proliferation, we investigated cell cycle distribution patterns using flow cytometry. Phosphorylation statuses of Erk, Jnk, p38, Akt as well as p53 and Cyclin D expression level were investigated by Western blotting.

Both 5-FU and ABL inhibited RPE cell proliferation. Only ABL promoted cell adhesion towards collagen I in hRPE3 cells. ABL was found to attenuate the rate of cell migration. Cell-mediated collagen gel contraction was attenuated by 5-FU only. Complementary inhibition in cellular proliferation and cell-mediated collagen gel contraction was observed when both 5-FU and ABL were applied. No significant cell death was observed after treatment with 5-FU, ABL or both.

ABL was found to reduce the amount of cells present at S phase. Akt and Erk were found to be hypo-phosphorylated and hyper-phosphorylated respectively after ABL treatment. The expression levels of phosphorylated-Jnk, phosphorylated-p38, p53, and Cyclin D1 were not altered when compared with the control.

These results showed that 5-FU and ABL complement with each other on inhibiting the wound healing activities of RPE cells in vitro without apparent cytotoxicity. They suggested a possible new treatment modality for PVR. ABL hypo-phosphorylated Akt and this observation is in line with the fact that ABL could attenuate cell proliferation.