Caspase-8 deficiency induces a switch from TLR3-induced apoptosis to lysosomal cell death in neuroblastoma cell lines
Evasion of apoptosis, one of the hallmarks of cancer, leads to anti-cancer therapy resistance. In order to overcome this resistance alternative forms of programmed cell death such as autophagic/lysosomal cell death and necrosis can be induced. TLR3 are inflammatory receptors expressed by both immune and epithelial cells. Their synthetic ligands are already used as adjuvant in cancer therapy, due to their role in modulating inflammatory signalling pathways.
Knowledge to date suggested that TLR3 functions via two mechanisms of action: (1) by facilitating the activation of caspase-8 and thus, the activation of the extrinsic apoptosis caspase-cascade, or (2) by recruiting and activating the key necrotic factor RIPK3, leading to plasma membrane permeabilization. In this article, Locquet and colleagues unveil a new mechanism of TLR3-induced cell death, executed by lysosomal cathepsins and accompanied by the enlargement and accumulation of lysosomes, which eventually result in the activation of the mitochondrial apoptotic pathway. This mechanism of lysosomal cell death is a default death mechanism in the absence of caspase-8, when the extrinsic apoptotic pathway is not activated .
To prove this, two neuroblastoma cell lines were cultured, and TLR3-induced lysosomal cell death was experimentally induced. Immunochemistry, gene quantification and cell viability studies were performed. An epifluorescent marker that is specific to lysosomes was then applied to the cell cultures and Nanolive’s 3D Cell Explorer-Fluo was used to record a time series of the 3D refractive index maps, with the corresponding lysosome-specific fluorescence signal. Lysosome observations allowed the authors to qualitatively account for increases in both the number and size of lysosomes (see Figure 1). Cells exposed to TLR3 were rounder and showed signs of membrane blebbing; both characteristic features of apoptosis. These results suggest that activation of the TLR3 induced caspase-independent lysosomal membrane permeabilization occurs at the same time as caspase activation downstream of lysosomal membrane permeabilization, resulting in an apoptotic morphology of the dying cell.
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Figure 1. IFN-1/poly(I:C) induced cell death involves lysosomes. Time series of SH-SY5Y cells 2D Refractive index I maps, individual lysosome-specific fluorescence signal (LysotrackerGreen™), and overlay of Lysosome segmentations after treatment with IFN-1/poly(I:C) (OA) (related to movie shown in Supplemental information (from Figure 3C in ).
 Locquet, Marie & Ichim, Gabriel & Dutour, Aurelie & Lebecque, Serge & Castets, Marie & Weber, Kathrin. (2020). Caspase-8 deficiency induces a switch from TLR3 induced apoptosis to lysosomal cell death in neuroblastoma cell lines. 10.1101/2020.01.13.904987.
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