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Tengku A. Kamalden, Anthony J. Bron, Neville N. Osborne; Rgc-5 Cell Death Caused By Serum Deprivation And Rotenone Occur By Different Processes And Only The Latter Is Blunted By Green Tea Flavonoids. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3094.
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To deduce how RGC-5 cells (cell line with some ganglion cell properties) die because of exposure to the mitochondrial complex 1 inhibitor rotenone or to a lack of serum and whether both modes of cell death are affected by the green tea flavonoids, epicatechin gallate (EC) and/or epigallacatechin gallate (EGCG).
Equivalent amounts of RGC-5 cells in 96, 24 or 6-well plates were maintained in culture for 24 hours. Thereafter, different concentrations of rotenone were added or the culture medium replaced with serum free culture medium. After 24-48 hours cells were analysed for their viability (MTT assay), reactive oxygen species (using the dye DHE) and for apoptosis (TUNEL). Proteins were extracted from samples after 24-96 hours and subjected to electrophoresis/western blotting.
Rotenone dose-dependently, and serum deprivation time-dependently, stimulated the production of ROS, increased staining for apoptosis (TUNEL) and reduced RGC-5 cell viability. Equivalent loss of RGC-5 cells (30-40%) was induced with 10µM of rotenone for 24 hours or by serum deprivation over 48 hours. Both EC and EGCG attenuated rotenone-induced cell death but neither substance affected serum deprivation-induced cell death. Significantly, western blot analysis showed both rotenone and serum deprivation to activate the p38 pathway. In contrast, the JNK pathway was only up-regulated by rotenone.
RGC-5 cell death-induced by serum deprivation or by inhibition of mitochondrial function with rotenone occur by different mechanisms with only the latter being blunted by EC and EGCG. These studies show that establishing candidate neuroprotectants for the treatment of a disease like glaucoma will not be easy should individual ganglion cells be triggered to die by different insults.
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