Trichostatin A induces cell death at the concentration recommended to differentiate the RGC-5 cell line

Supplementation with Trichostatin A (TSA) has been described as the method of choice for differentiating the RGC-5 cell line into cells with neuronal properties. However, TSA is known to induce apoptosis. We therefore investigated whether TSA at the recommended concentration for differentiation (500 nM) and at three additional concentrations (40, 150 and 2000 nM) induces apoptosis or cell death in the RGC-5 cell line. Morphological changes of the RGC-5 cells occurred after 24 and 48 hours (h) of treatment with 500 and 2000 nM TSA. Differentiation of RGC-5 cell began at 150 nM. A decrease in the cell count was observed from 150 nM TSA onwards compared to controls. Five hundred nanomolar of TSA reduced the amount of cells to 51% (p<0.005) after 24h and to 24% (p<0.005) after 48 h compared to controls on crystal violet staining. At 500 nM TSA a massive induction of apoptosis after 24 and 48 h was noted. Supplementation of 500 nM TSA increased caspase 3/7 activity 5.0-fold (p<0.005). Furthermore, 27× more TUNEL-positive cells were found and the cleaved caspase 3/caspase 3 ratio was 1.8-fold (p<0.1) higher 24h after the addition of 500 nM TSA. The Bax/Bcl-2 ratio was 3.4-fold (p<0.05) higher after 48 h. Cell viability decreased to 70% (p<0.005) and to 35% (p<0.005) after 24 and 48 h, respectively. Moreover, 103× (p<0.05) more dead cells (via propidium iodide staining) were found after 48 h of treatment with 500 nM TSA. In conclusion, TSA induces cell death and apoptosis at the concentration recommended for differentiation. The induction of apoptosis occurred dose and time dependently and already at even lower concentrations of TSA which did not lead to differentiation induced apoptosis. Thus, studies with RGC-5 cells should not be performed within the first 48 h after supplementation with TSA.