PURPOSE. Lectin from the edible mushroom Agaricus bisporus (ABL) was found to inhibit cell proliferation of some ocular and cancer cell lines. To elucidate how ABL inhibited RPE cell proliferation, we investigated the changes in cell cycle distribution and cell proliferation-related signaling pathways after ABL treatment.METHODS. Primary human RPE cells were isolated and grown in DMEM/F12 with or without the ABL (20 or 90 lg/mL) for 3 days. Analysis of cell cycle was performed by flow cytometry. Phosphorylation status of Erk, Jnk, p38, and Akt as well as p53 expression levels were investigated by Western blotting. The role of phosphorylated-Akt in RPE cell proliferation was further evaluated using LY294002.
RESULTS.After ABL treatment (90 lg/mL), the amount of cells present in the S phase was found to be reduced. These changes were not apparent in cells treated with 20 lg/mL ABL. In addition, Erk and Akt were found to be hyperphosphorylated and hypophosphorylated, respectively. The expression levels of phosphorylated-Jnk, phosphorylated-p38, and p53 were not altered when compared with those of the control cells. When RPE cells were treated with LY294002 and deprived from phosphorylated-Akt expression, cell proliferation rate was reduced. Reduction in the amount of cells present in S phase was also observed.
CONCLUSIONS.Our results showed that ABL hypophosphorylated Akt and this observation is in line with the fact that ABL attenuates cell proliferation. As the level of p53 was not significantly altered by ABL, this indicated that ABL-arrested cell cycle progression was independent of p53 activation. (Invest Ophthalmol Vis Sci. 2012; 53:7469-7475)
Anomalous retinal pigment epithelial (RPE) cells have been implicated in the development of retinal diseases. Lectin from the edible mushroom Agaricus bisporus (ABL) was found to inhibit growth of RPE cells. To elucidate the mechanism through which ABL inhibits RPE cell proliferation, we investigated the changes in cell proliferation-related signaling pathways and cell cycle distribution patterns. Primary human RPE cells were grown with or without the lectin (ABL) supplement (20ug or 90ug/ml) for three days. Phosphorylation statuses of Akt, Jnk and p38 as well as p53 expression level were investigated by Western blotting. Cellular distributions in various cell cycle phases were investigated using flow cytometry. After ABL treatment (90ug/ml), Akt was found to be hypo-phosphorylated while the expression levels of p53, phosphorylated-Jnk and phosphorylated-p38 were not altered. The amount of cells present at S phase was reduced. Our results showed that ABL hypo-phosphorylated Akt and this observation is in line with the finding that ABL could attenuate cell proliferation. As the level of p53 was not significantly altered by ABL, this suggested that the mechanism in which ABL arrested cell proliferation was independent of Akt-mediated MDM2 activation but was possibly mediated by altering G1 to S phase transition.
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