Objective To investigate the enhancement of eucommia chlorogenic acid(CGA) on the radiosensitivity of retinoblastoma cell line HXO-Rb44 by regulating PI3K/AKT/Nrf-2 pathway. Methods HXO-Rb44 cells were treated with different concentrations of CGA, then IC10 was detected as CGA concentration in subsequent experiments. Cells were divided into 4 groups: a Ctrl group, a Radio group, a CGA group and a Radio+CGA group. Radio group was disposed with 4 MV X-rays for 24 h, CGA group was processed with CGA for 24 h, and Radio+CGA group was disposed with 4 MV X-rays for 24 h after CGA treatment for 24 h. Flow cytometry was used to detect the cell cycle and cell apoptosis. The protein expressions of Ki67, NAD (P) H quinone oxidoreductase I (NQO1), cleaved-caspase-3 (cl-caspase-3), thioredoxin reductase-1 (TrxR1), activated phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), p-AKT and nuclear factor-erythroid 2-related factor 2 (Nrf2) were determined by Western blot. Furthermore, PI3K activator 740Y-P was added to detect, the cell cycle, cell apoptosis, cell proliferation and apoptosis associated protein expressions, expressions of proteins in PI3K/AKT/Nrf2 pathway. Results With the increasing concentrations of CGA, the relative survival rates of cells were decreased, and the drug toxicity was concentration-dependent. The IC10 value was 81.59 μmol/L. Compared with Ctrl group, the cell population in G2/M period was significantly increased, and the cell apoptosis rates were increased in Radio group and CGA group (P<0.01). Compared with Radio group, the cell population in G2/M period and cell apoptosis rates were increased further in Radio+CGA group. Meanwhile, compared with Ctrl group, the protein expressions of Ki67, NQO1, TrxR1, p-PI3K, Nrf2 and p-AKT/AKT ratios in other groups were decreased significantly (P<0.01), and the combined effect of Radiation and CGA further reduced the above indicators in Radio+CGA group. Furthermore, PI3K activator could reverse the effects of radiation treatment on cell cycle, cell proliferation, cell apoptosis and PI3K/AKT/Nrf2 pathway. CGA could restore the changes of the above indicators caused by radiation (P<0.01). Conclusions CGA can enhance the radiosensitivity of retinoblastoma cell line HXO-Rb44, and the protective effect of CGA may be related to its inhibition of PI3K/AKT/Nrf2 pathway.
Key words
Eucommia chlorogenic acid; /
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Radiosensitivity; /
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Retinoblastoma; /
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PI3K/AKT/Nrf2 pathway
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