Regulated cell death (RCD) in cancer: key pathways and targeted therapies.

Regulated cell death (RCD), also well-known as programmed cell death (PCD), refers to the form of cell death that can be regulated by a variety of biomacromolecules, which is distinctive from accidental cell death (ACD). Accumulating evidence has revealed that RCD subroutines are the key features of tumorigenesis, which may ultimately lead to the establishment of different potential therapeutic strategies. Hitherto, targeting the subroutines of RCD with pharmacological small-molecule compounds has been emerging as a promising therapeutic avenue, which has rapidly progressed in many types of human cancers. Thus, in this review, we focus on summarizing not only the key apoptotic and autophagy-dependent cell death signaling pathways, but the crucial pathways of other RCD subroutines, including necroptosis, pyroptosis, ferroptosis, parthanatos, entosis, NETosis and lysosome-dependent cell death (LCD) in cancer. Moreover, we further discuss the current situation of several small-molecule compounds targeting the different RCD subroutines to improve cancer treatment, such as single-target, dual or multiple-target small-molecule compounds, drug combinations, and some new emerging therapeutic strategies that would together shed new light on future directions to attack cancer cell vulnerabilities with small-molecule drugs targeting RCD for therapeutic purposes.

Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate.

Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.

The Antifungal Protein AfpB Induces Regulated Cell Death in Its Parental Fungus Penicillium digitatum.

Filamentous fungi produce small cysteine-rich proteins with potent, specific antifungal activity, offering the potential to fight fungal infections that severely threaten human health and food safety and security. The genome of the citrus postharvest fungal pathogen Penicillium digitatum encodes one of these antifungal proteins, namely AfpB. Biotechnologically produced AfpB inhibited the growth of major pathogenic fungi at minimal concentrations, surprisingly including its parental fungus, and conferred protection to crop plants against fungal infections. This study reports an in-depth characterization of the AfpB mechanism of action, showing that it is a cell-penetrating protein that triggers a regulated cell death program in the target fungus. We prove the importance of AfpB interaction with the fungal cell wall to exert its killing activity, for which protein mannosylation is required. We also show that the potent activity of AfpB correlates with its rapid and efficient uptake by fungal cells through an energy-dependent process. Once internalized, AfpB induces a transcriptional reprogramming signaled by reactive oxygen species that ends in cell death. Our data show that AfpB activates a self-injury program, suggesting that this protein has a biological function in the parental fungus beyond defense against competitors, presumably more related to regulation of the fungal population. Our results demonstrate that this protein is a potent antifungal that acts through various targets to kill fungal cells through a regulated process, making AfpB a promising compound for the development of novel biofungicides with multiple fields of application in crop and postharvest protection, food preservation, and medical therapies.IMPORTANCE Disease-causing fungi pose a serious threat to human health and food safety and security. The limited number of licensed antifungals, together with the emergence of pathogenic fungi with multiple resistance to available antifungals, represents a serious challenge for medicine and agriculture. Therefore, there is an urgent need for new compounds with high fungal specificity and novel antifungal mechanisms. Antifungal proteins in general, and AfpB from Penicillium digitatum in particular, are promising molecules for the development of novel antifungals. This study on AfpB's mode of action demonstrates its potent, specific fungicidal activity through the interaction with multiple targets, presumably reducing the risk of evolving fungal resistance, and through a regulated cell death process, uncovering this protein as an excellent candidate for a novel biofungicide. The in-depth knowledge on AfpB mechanistic function presented in this work is important to guide its possible future clinical and agricultural applications.

A Flow Cytometry-Based Assay for High-Throughput Detection and Quantification of Neutrophil Extracellular Traps in Mixed Cell Populations.

Neutrophil extracellular traps (NETs) are web-like structures composed of decondensed chromatin and antimicrobial proteins that are released into the extracellular space during microbial infections. This active cell death program is known as NETosis. To date, florescence microscopy is the widely accepted method for visualization and quantification of NETs. However, this method is subjective, time consuming and yields low numbers of analyzed polymorphonuclear cells (PMNs) per sample. Increasing interest has emerged on the identification of NETs using flow cytometry techniques. However, flow cytometry analysis of NETs requires particular precautions for sample preparation to obtain reproducible data. Herein, we describe a flow cytometry-based assay for high-throughput detection and quantification of NETosis in mixed cell populations. We used fluorescent-labeled antibodies against cell markers on PMNs together with a combination of nucleic acid stains to measure NETosis in whole blood (WB) and purified PMNs. Using plasma membrane-impermeable DNA-binding dye, SYTOX Orange (SO), we found that cell-appendant DNA of NETting PMNs were positive for SO and DAPI. The combination of optimally diluted antibody and nucleic acid dyes required no washing and yielded low background fluorescence. Significant correlations were found for NETosis from WB and purified PMNs. We then validated the assay by comparing with time-lapse live cell fluorescence microscopy and determined very good intraassay and interassay variances. The assay was then applied to a disease associated with NETosis, systemic lupus erythematosus (SLE). We examined PMA-induced NETosis in peripheral PMNs from SLE patients and controls and in bone marrow PMNs from multiple murine models. In summary, this assay is observer-independent and allows for rapid assessment of a large number of PMNs per sample. Use of this assay does not require sophisticated microscopic equipment like imaging flow cytometers and may be a starting point to analyze extracellular trap formation from immune cells other than PMNs. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.