Abstract
Dysregulated expression of Fas-associated death domain (FADD) is associated with the impediment of various cellular pathways, including apoptosis and inflammation. The adequate cytosolic expression of FADD is critical to the regulation of cancer cell proliferation. Importantly, cancer cells devise mechanisms to suppress FADD expression and, in turn, escape from apoptosis signaling. Formulating strategies, for direct delivery of FADD proteins into cancer cells in a controlled manner, may represent a promising therapeutic approach in cancer therapy. We chemically conjugated purified FADD protein with cell permeable TAT (transactivator of transcription) peptide, to deliver in cancer cells. TAT-conjugated FADD protein internalized through the caveolar pathway of endocytosis and retained in the cytosol to augment cell death. Inside cancer cells, TAT-FADD rapidly constituted DISC (death inducing signaling complex) assembly, which in turn, instigate apoptosis signaling. The apoptotic competency of TAT-FADD showed comparable outcomes with the conventional apoptosis inducers. Notably, TAT-FADD mitigates constitutive NF-κB activation and associated downstream anti-apoptotic genes Bcl2, cFLIPL, RIP1, and cIAP2, independent of pro-cancerous TNF-α priming. In cancer cells, TAT-FADD suppresses the canonical NLRP3 inflammasome priming and restricts the processing and secretion of proinflammatory IL-1β. Our results demonstrate that TAT-mediated intracellular delivery of FADD protein can potentially recite apoptosis signaling with simultaneous regulation of anti-apoptotic and proinflammatory NF-κB signaling activation in cancer cells.
Highlights
The Fas-associated death domain (FADD) protein orchestrates several cellular pathways, including apoptosis, cell cycle regulation, autophagy, and inflammation [1,2]
Post-translational modifications (PTMs) and nuclear localization of FADD have been reported in cancer cells, which further challenge the pro-apoptotic competency of FADD to instigate apoptosis signaling [9,10,11]
(A) Schematic diagram representing the target site of proposed apoptosis inducers. (B–E) HCT 116 cells were treated with CD 95L (200 ng/mL), tumor necrosis factor-α (TNF-α) (50 ng/mL), etoposide (50 μM), HA14-1 (5 μM), protein translational inhibitor cycloheximide (CHX, 5 μg/mL), and TAT-FADD (5 μM) alone for the mentioned time points, (B) The bright field images of cells counterstained with DAPI, post treatments, representative of 150 cells from 3 independent fields, scale bar 5 μm, (C) % apoptotic death by a TaliTM image-based cytometer, (D) % change in MMP and (E) expression of Procaspase-7 and cleavage of PARP by Western blot analysis, molecular weight marker left to each blot
Summary
The Fas-associated death domain (FADD) protein orchestrates several cellular pathways, including apoptosis, cell cycle regulation, autophagy, and inflammation [1,2]. Pathogen recognition receptor (PRR)-mediated NF-κB activation induces transcriptional priming of proinflammatory genes to maintain the tumor microenvironment [25,26] In this context, the NLRP3 inflammasome complex consisting of NLRP3 (nucleotide-binding domain leucine-rich repeat (NLR) and pyrin domain containing receptor 3), ASC (Apoptosis-associated speck-like protein containing a CARD), and pro-caspase-1 protein facilitates the proteolytic processing of NF-κB-induced proinflammatoryIL-1β to promote tumor growth [27,28,29]. This study provides a novel insight of the molecular mechanism and delivery of FADD protein, which can target cancer cell proliferation and NF-κB activation with subsequent suppression of pro-tumorigenic and proinflammatory signaling. This indispensable approach may be beneficial to the consideration of protein as a drug candidate in the revolution of anticancer therapy
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