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Sven Schnichels, Maximilian Schultheiss, Johanna Hofmann, Peter Szurman, Karl U. Bartz-Schmidt, Martin S. Spitzer; Trichostatin A Induces Cell Death At The Concentration Recommended To Differentiate The RGC-5 Cell Line. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4595.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate whether Trichostatin A (TSA) at the recommended concentration for (re-) differentiation and three additional concentrations induces apoptosis in the retinal ganglion cell (RGC)-5 cell line.
40nM, 150nM, 500nM or 2000nM of TSA were supplemented on RGC-5 cells for 1, 24 and 48 hours. Cell morphology and cell death, via propidium iodide (PI) staining, were investigated with phase contrast and fluorescence microscopy, respectively. Cell amount and cell viability were analyzed by crystal violet staining and MTS assay, respectively. Apoptosis was detected by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), analyzing caspase 3/7 activity, determining cleaved caspase 3/caspase 3 and BAX/Bcl-2 ratio via Western blot.
Morphological changes of the RGC-5 cells occurred 24h and 48h after treatment with 500nM and 2000nM TSA. Almost all cells at 150nM TSA were unchanged. Obviously fewer cells were observable at 150 nM, 500nM and 2000nM TSA compared to controls. These findings were confirmed by crystal violet staining, which showed that 500nM TSA supplementation reduced the amount of cells to 51% (p<0.0005) 24h after treatment and to 24% (p<0.0005) 48h after treatment compared to controls. We found a massive induction of apoptosis at the recommended doses 24h and 48h after treatment compared to controls: supplementation of 500nM TSA increased caspase 3/7 activity 24h after treatment by 5.0-fold (p<0.0005). Moreover, 27x (p<0.0005) more TUNEL-positive cells were found 24h after treatment with 500nM TSA. The cleaved caspase 3/caspase 3 ratio was 1.8-fold (p<0.05) higher 24h after 500nM TSA supplementation, and the BAX/Bcl-2 ratio was 3.4-fold (p<0.05) higher 48h after treatment. In parallel, cell viability decreased to 70% (p<0.0005) 24h and to 35% (p<0.0005) 48h after treatment with 500nM TSA. Even more, 103x (p<0.0005) more PI-positive cells were found 48h after treatment with 500nM TSA. All methods showed a dose dependent effect of TSA. However, no significant changes were found 1h after treatment.
TSA induces cell death / apoptosis at the concentration recommended for (re-) differentiation. Furthermore, the induction of cell death occurred dose-dependently and even lower concentrations of TSA which did not lead to (re-)differentiation induced cell death.
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