All-trans retinoic acid-induced apoptosis signaling pathway in ARPE-19 cells

Objective

To investigate all-trans retinoic acid(ATRA)-induced apoptosis signaling pathway in ARPE-19 cells in vitro.

Methods

The APRE-19 cell was treated with different concentrations of ATRA for 24 hours and 48 hours.Cell counting kit-8 (CCK-8) was used to detect the cell viability in order to determine the experimental concentration range.Flow cytometry and Western blot method were performed to evaluate the apoptosis and caspase related protein levels in ARPE-19 cells treated with 0, 2.5, 5, 10, 15 and 20 μmol/L of ATRA for 24 hours.Flow cytometry was used to detect the reactive oxygen species (ROS) and multicaspase levels and quantitative real-time PCR was carried out to determine the mRNA relative expression levels of caspase related proteins in ARPE-19 cells treated with 0, 2.5, 5, 10 and 20 μmol/L of ATRA, and 0 μmol/L ATRA group was used as the blank control group.

Results

CCK-8 test showed that the half maximal inhibitory concentration of ARPE-19 cells treated with different concentrations of ATRA for 24 hours and 48 hours were 13.88 μmol/L and 11.99 μmol/L, respectively.The cell survival rates of ARPE-19 cells treated with different concentrations of ATRA for 24 hours and 48 hours were significantly different (F=176.60, 350.30; both at P<0.01). When cultured for 24 hours, the cell survival rates of ARPE-19 cells in the 2 μmol/L and 6 μmol/L of ATRA groups were higher than that of the blank control group (both at P<0.05), and the cell survival rates of ARPE-19 cells in the 12, 14, 16, 18 and 20 μmol/L of ATRA groups were lower than that of the blank control group (all at P<0.05). Flow cytometry showed that there were significant differences in the apoptosis, ROS and multicaspase level among ARPE-19 cell groups treated with different concentrations of ATRA (F=86.39, 116.84, 101.40; all at P<0.01). The apoptosis rates of APRE-19 cells in the 2.5 μmol/L and 5 μmol/L of ATRA groups were significantly decreased than that of the blank control group, and the apoptosis rate of APRE-19 cells in the 10, 15 and 20 μmol/L of ATRA groups were significantly increaseded than that of the blank control group (all at P<0.01). The relative expression levels of multicaspase and ROS were significantly higher in the 2.5, 5, 10 and 20 μmol/L of ATRA groups than that of the blank control group (all at P<0.01). Western blot assay showed that the relative expression level of caspase 9 was increased in the 2.5, 5, 10, 15 and 20 μmol/L of ATRA groups than that of the blank control group (all at P<0.05). Compared with the blank control group, the relative expression levels of caspase 12 were increased in the 2.5 μmol/L of ATRA group and reduced gradually in the 5, 10, 15 and 20 μmol/L of ATRA groups, among which there were significant differences between the blank control group and 2.5, 15, and 20 μmol/L of ATRA groups (all at P<0.05). The relative expression level of caspase 3 was significantly increased in the 5, 10 and 20 μmol/L of ATRA groups than that of the blank control group (all at P<0.05). The relative expression level of cleaved caspase 3 was significantly increased in the 2.5, 5, 10, 15 and 20 μmol/L of ATRA groups than that of the blank control group (all at P<0.01). Quantitative real-time PCR assay showed that the relative expression levels of caspase 9 and caspase 12 mRNA were significantly higher in the 2.5, 5, 10 and 20 μmol/L of ATRA groups than that of the blank control group (all at P<0.01). The relative expression levels of caspase 3 mRNA were significantly higher in the 5 μmol/L and 10 μmol/L of ATRA groups than that of the blank control group (both at P<0.01). When the concentration of ATRA was lower than 10 μmol/L, the relative expression levels of caspase 9 and caspase 12 mRNA were elevated in a concentration-dependent manner.When the concentration of ATRA reached 20 μmol/L, the relative expression levels of caspase 9 and caspase 12 mRNA were markedly decreased, but it was still higher than that of the blank control group.

Conclusions

ATRA induces apoptosis in ARPE-19 cells in vitro through activating the reactive oxygen species and endogenous caspase-dependent apoptotic pathway.

All-trans retinoic acid; Retinal pigment epithelial cells; Response to oxidative stress; Caspase apoptotic pathway