Figure 5.

Inhibition of cell death pathways decreases tamoxifen-mediated ARPE-19 cytotoxicity. (A) Treatment of tamoxifen-exposed ARPE-19 cells with inhibitors for Nec-1, caspase-1, caspase-3, or cathepsin B/L significantly reduced cell death (as measured by LDH release). Combining caspase or cathepsin inhibitors with Nec-1 further significantly reduced tamoxifen-mediated cell death, as compared to treatment with caspase and cathepsin inhibitors alone. (B) Abnormal morphology of ARPE-19 cells was observed after tamoxifen administration, with rounded cells and patches of cell loss visible (top left). Treatment of ARPE-19 cells with Nec-1 (bottom row, first section) and pan-caspase inhibitors (top row, second section) alone inhibited cell death, but cellular morphology appeared abnormal, suggesting cell distress. Caspase-1 inhibitor treatment alone partially rescued cell death; however, abnormal cellular morphology was still observed (top row, third section). Combinatorial treatment of Nec-1 with pan-caspase inhibitor (bottom row, second panel) or Nec-1 with caspase-1 inhibitor (bottom row, third section) resulted in near complete inhibition of cell death and preservation of normal morphology (as shown in Fig. 1C). Scale bar: 200 μm. (C) The LDH assay reveals that inhibition using a combined cathepsin B/L inhibitor significantly reduced LDH release. Inhibition of cathepsin B or cathepsin L alone shows moderate reduction of LDH release in tamoxifen-induced ARPE-19 cytotoxicity when compared with the controls. Concurrent inhibition with individual cathepsin B and cathepsin L inhibitors drastically reduced LDH release with tamoxifen treatment, similar to use of the combined cathepsin B/L inhibitor. (D) Inhibition of caspase-9, but not caspase-8, significantly reduced LDH release in tamoxifen-treated ARPE-19 cells. Inhibition of caspase-8 together with Nec-1 or inhibition of caspase-9 along with Nec-1 dramatically reduced LDH release. Error bars: ±SEM. Adjusted P values: **P < 0.05, ***P < 0.005, ****P < 0.0001.