Antiproliferative and apoptotic effects of indomethacin on human retinoblastoma cell line Y79 and the involvement of β-catenin, nuclear factor-κB and Akt signaling pathways.

BACKGROUND: To determine in vitro if indomethacin inhibits proliferation and induces apoptosis in human retinoblastoma cell line Y79, and to explore possibly involved signaling pathways.
METHODS: The human retinoblastoma cell line Y79 was cultured with indomethacin at various concentrations (0, 25, 50, 100, 200 and 400 µmol/l). The effect of indomethacin on cell proliferation and apoptosis was examined by the Cell Counting Kit-8 and TUNEL test, respectively. The mRNA level of survivin, β-catenin and Bcl-2 was detected by RT-PCR. The protein level of survivin was measured by ELISA. Western blot was used to analyze β-catenin, nuclear factor (NF)-κB/p65, phosphorylated Akt (pAkt) and total Akt (tAkt) expression in cultured cells.
RESULTS: Indomethacin treatment inhibits proliferation (at concentrations from 25 to 400 µmol/l) and induces apoptosis (at concentrations from 100 to 400 µmol/l) of human retinoblastoma cell line Y79 in a dose-dependent manner. RT-PCR showed that the mRNA expression of Bcl-2 (F = 20.497; p < 0.001) and of β-catenin (F = 14.835; p < 0.001) was significantly different among the treated groups. Survivin mRNA levels remained steady, but its protein levels decreased significantly as measured by ELISA (F = 67.633; p < 0.001). Western blot analysis showed a dose-dependent downregulation of β-catenin (F = 37.411; p < 0.001), NF-κB/p65 (F = 16.302; p < 0.001) and of pAkt (F = 27.700; p < 0.001) after indomethacin treatment, while tAkt protein expression was steady among the groups.
CONCLUSIONS: Treatment with indomethacin can potently suppress proliferation and induce apoptosis in the retinoblastoma Y79 cell line. Wnt/β-catenin, NF-κB and Akt/PKB pathways might be implicated in the process.