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T. A. Tengku Kamalden, A. J. Bron, N. N. Osborne; RGC-5 Cell Apoptosis Caused by Rotenone and White Light Are Similar but Not Identical With Both Being Attenuated by Two Green Tea Flavonoids. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4519.
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To compare the mechanism by which rotenone and light kills transformed cells with certain ganglion cell properties (RGC-5 cells) and to compare the protective effects of two naturally occurring flavonoids, epicatechin gallate (ECG) and epigallocatechin gallate (EGCG).
Equivalent amounts of RGC-5 cells were transferred to 96- or 12-well plates and exposed to different concentrations of rotenone or to white light (400-700nm) alone or in combination with defined substances provided 60 minutes earlier. Cells were analysed 24-96 hours later for their viability (MTT procedure), production of reactive oxygen species (ROS) using DHE and occurrence of apoptosis (staining for phosphatidylserine). Moreover, proteins were extracted and subjected to electrophoresis and western blotting for various proteins involved in cell death.
Rotenone like light, increased ROS production dose-dependently and reduced the viability of RGC-5 cell survival. Rotenone (10µM) and light-induced cell death was by apoptosis indicated by positive staining for phosphatidylserine and was attenuated by PARP and necroptosis inhibitors (necrostatin-1). Both rotenone and light stimulated p-c-Jun, heme-oxygenase-1 and PARP proteins, while only light insult differentially cleaved AIF protein. Rotenone and light-induced apoptosis were equally counteracted by EGCG and ECG.
Rotenone causes apoptosis by inhibition of mitochondrial complex I while the effect of light is mitochondrial-dependent. The present results show that the mechanisms by which rotenone and light induce cell death are not identical. However, both mechanisms are attenuated in a similar way by EGCG and ECG.
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