Intracellular Death Pathways Research Articles

Pyroptosis-Inducing Biomaterials Pave the Way for Transformative Antitumor Immunotherapy.

Pyroptosis can effectively overcome immunosuppression and reactivate antitumor immunity. However, pyroptosis initiation is challenging. First, the underlying biological mechanisms of pyroptosis are complex, and a variety of gasdermin family proteins can be targeted to induce pyroptosis. Second, other intracellular death pathways may also interfere with pyroptosis. The rationally designed gasdermin protein-targeting biomaterials are capable of inducing pyroptosis and have the capacity to stimulate antitumor immune function in a safe and effective manner. This review provides a comprehensive overview of the design, function, and antitumor efficacy of pyroptosis-inducing materials and the associated challenges, with a particular focus on the design options for pyroptosis-inducing biomaterials based on the activation of different gasdermin proteins. This review offers a valuable foundation for the further development of pyroptosis-inducing biomaterials for clinical applications.

Abstract CT179: Safety and effectiveness of plasmapheresis-based elimination of soluble TNFα receptors combined with chemotherapy in advanced, chemorefractory triple-negative breast cancer patients - a phase I/II study (CP7-005)

Abstract Introduction: Tumor necrosis factor-alpha (TNFα) is a pleiotropic cytokine with known antitumor activity, produced mainly by activated immune cells. Cancer cells neutralize TNFα by shedding soluble TNF receptors 1&2 (sTNF-Rs), which act as TNFα-binding decoys, promoting cancer cell proliferation, survival, and chemoresistance. Immunopheresis® employs therapeutic apheresis with the selective immunoadsorption LW-02 column (LW-02) for treating solid malignancies. LW-02-based Immunopheresis (granted FDA Breakthrough Device Designation and a CE Mark for mTNBC) selectively removes sTNF-Rs from plasma, permitting TNFα to bind to membrane-bound TNF-Rs, activating intracellular death pathways, and also modulate T-cell-mediated immune activity. Part A data of our phase I/II clinical trial in metastatic, chemorefractory, triple-negative breast cancer (mTNBC) patients (NCT04004910) confirmed that LW-02-based Immunopheresis monotherapy is safe and well-tolerated, with signs of disease stabilization (SD) in patients treated >4 weeks. Here we present interim data on the safety and preliminary efficacy of LW-02 Immunopheresis combined with chemotherapy. Methods: LW-02 Immunopheresis (3x/week for 16 weeks) is combined with a weekly paclitaxel (80 mg/m2)+carboplatin (AUC 2) regimen in patients with mTNBC; treatment is continued past 16 weeks in clinical and/or objective responders. Primary endpoints are safety and tolerability. Secondary endpoints assessed in patients treated >4 weeks include overall survival, tumor response according to RECIST, rate of CNS progression and quality-of-life. Results: Of 11 patients enrolled in Part B of the study, 7 patients were treated for >4 weeks. The ORR was 18% (1CR, 1PR) with one additional patient experiencing SD; the ORR and CBR in patients treated >4 weeks were 29% and 43%, respectively. The median OS, to date, is 18.6 weeks and 26.7 weeks in the group treated >4 weeks. The rate of CNS progression (new or preexisting lesions) appears lower than expected. The most common adverse events (AEs) were chemotherapy-induced myelotoxicity (anemia, neutropenia, thrombocytopenia) and transient electrolyte abnormalities. Thirty-one serious AEs (SAEs) were reported for all 11 patients, 15 (48.4%) were transfusion requiring anemias. Of the SAEs, 3 (9.6%) were judged to have a potential causal relationship to LW-02 column Immunopheresis. Conclusion: LW-02-based Immunopheresis combined with weekly chemotherapy is generally safe, well-tolerated and highly effective in specific sTNFR subtraction on a longer-term basis, with promising signals of clinical benefit in heavily pretreated mTNBC patients (median 3.3 [2-5] prior lines of systemic therapy). Further clinical evaluation of the antitumor activity of LW-02-based immunopheresis combined with low-dose chemotherapy is ongoing with a focus on quality-of-life and prevention of CNS disease progression, the latter especially important in chemorefractory mTNBC, given the high prevalence of CNS involvement. Citation Format: Piotr Jan Wysocki, Adam Ostrowski, Robert Segal, Victoria Manax, Pawel Potocki, Kamil Konopka, Lukasz Kwinta, Paulina Fraczek, Maciej Lubas, Mateusz Lobacz, Lawrence Florin. Safety and effectiveness of plasmapheresis-based elimination of soluble TNFα receptors combined with chemotherapy in advanced, chemorefractory triple-negative breast cancer patients - a phase I/II study (CP7-005) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT179.

A mitochondrial targeting tetrapeptide Bendavia protects lateral line hair cells from gentamicin exposure

The hearing loss induced by aminoglycosides is caused by the permanent loss of mechanosensory hair cells of the inner ear. The aim of the present study is therefore to evaluate the protective effect of Bendavia, a novel antioxidant, on gentamicin-induced hair cell damage in zebrafish lateral lines. The results demonstrated the pretreatment of Bendavia exhibited dose-dependent protection against gentamicin in both acute and chronic exposure. We found that Bendavia at 150μm conferred optimal protection from either acute or chronic exposure with ototoxin. Bendavia reduced uptake of fluorescent-tagged gentamicin via mechanoelectrical transduction channels, suggesting its protective effects may be partially due to decreasing ototoxic molecule uptake. The intracellular death pathways inhibition triggered by gentamicin might be also included as no blockage of gentamicin was observed. Our data suggest that Bendavia represents a novel otoprotective drug that might provide a therapeutic alternative for patients receiving aminoglycoside treatment.

Ischemic Neuronal Damage

Knowledge of the molecular mechanisms that underlie neuron death following stroke is important to allow the development of effective neuroprotective strategies. Since studies in human stroke are extremely limited due to the inability of collecting post mortem tissue at time points after the onset of stroke where neuronal death occurs, brain ischemia research focuses on information derived from animal models of ischemic injury. The two principal models for human stroke are induced in rodents either by global or focal ischemia. In both cases, blood flow disruptions limit the delivery of oxygen and glucose to neurons causing ATP reduction and energy depletion, initiating excitotoxic mechanisms that are deleterious for neurons. These include activation of glutamate receptors and release of excess glutamate in the extracellular space inducing neuron depolarisation and dramatic increase of intracellular calcium that in turn activates multiple intracellular death pathways. The notion that excitotoxicity leads only to neuron necrosis has been abandoned, as ultrastructural and biochemical analysis have shown signs of apoptotic and autophagic cell death in ischemic neurons and this has been further confirmed in neurons subjected to in vitro ischemia models. Both in vitro and in vivo studies, targeting a single death mechanism either by the inhibition of death-inducing molecules or the overexpression of antiapoptotic components in neurons, have shown tremendous neuroprotective potential. Despite their effectiveness in preclinical studies, a large number of neuroprotectants have failed in clinical trials for stroke suggesting that we still lack essential knowledge on the triggers and mediators of ischemic neuron death. In this review evidence will be presented on how ischemic injury occurs, what death mechanisms are activated and how these can be manipulated to induce neuroprotection.

Structural Features of Galectin-9 and Galectin-1 That Determine Distinct T Cell Death Pathways

The galectin family of lectins regulates multiple biologic functions, such as development, inflammation, immunity, and cancer. One common function of several galectins is the ability to trigger T cell death. However, differences among the death pathways triggered by various galectins with regard to glycoprotein receptors, intracellular death pathways, and target cell specificity are not well understood. Specifically, galectin-9 and galectin-1 both kill thymocytes, peripheral T cells, and T cell lines; however, we have found that galectin-9 and galectin-1 require different glycan ligands and glycoprotein receptors to trigger T cell death. The two galectins also utilize different intracellular death pathways, as galectin-9, but not galectin-1, T cell death was blocked by intracellular Bcl-2, whereas galectin-1, but not galectin-9, T cell death was blocked by intracellular galectin-3. Target cell susceptibility also differed between the two galectins, as galectin-9 and galectin-1 killed different subsets of murine thymocytes. To define structural features responsible for distinct activities of the tandem repeat galectin-9 and dimeric galectin-1, we created a series of bivalent constructs with galectin-9 and galectin-1 carbohydrate recognition domains connected by different peptide linkers. We found that the N-terminal carbohydrate recognition domain and linker peptide contributed to the potency of these constructs. However, we found that the C-terminal carbohydrate recognition domain was the primary determinant of receptor recognition, death pathway signaling, and target cell susceptibility. Thus, carbohydrate recognition domain specificity, presentation, and valency make distinct contributions to the specific effects of different galectins in initiating T cell death.

Structural Features of Galectin‐9 and Galectin‐1 that Determine Distinct T cell Death Pathways

Galectin‐9 and galectin‐1 both kill thymocytes, peripheral T cells and T cell lines. However, differences in target cell specificity, glycoprotein receptor requirements, and intracellular death pathways for these two galectins are not well understood. We found that galectin‐9 and galectin‐1 required different glycan ligands and glycoprotein receptors to trigger T cell death. They also triggered different intracellular death pathways, as only galectin‐9 T cell death was blocked by intracellular bcl‐2, while only galectin‐1 T cell death was blocked by intracellular galectin‐3. Galectin‐9 and galectin‐1 were both expressed in murine thymus, but killed different thymocyte subsets, with mature thymocytes more susceptible to galectin‐9. To define the structural features responsible for distinct activities of galectin‐1 and galectin‐9, we created a series of bivalent neo‐galectins with galectin‐1 and galectin‐9 carbohydrate recognition domains on different peptide linkers. The N‐terminal carbohydrate recognition domain and linker peptide contributed to potency of the galectins. Strikingly, the C‐terminal carbohydrate recognition domain determined receptor recognition, death pathway signaling and target cell susceptibility. Thus, carbohydrate recognition domain specificity, presentation, and valency all contribute to specific effects of different galectins in regulating T cell death.

Expression of ASK1 during chick and early mouse development

Apoptosis signal-regulating kinase 1 (ASK1) is an important regulator of stress-induced cell death. ASK1 is activated by oxidative stress, TNF and endoplasmatic reticulum stress and activates the JNK- and p38-dependent intracellular death pathways. A number of studies have suggested that ASK1 may also have other roles in addition to its pro-apoptotic activity. Expression of ASK1 during early embryonic development has so far not been analyzed. We have identified and cloned chick ASK1 in a screen for FGF8 inducible genes in chick facial mesenchyme. Here we report the expression of chick ASK1 from the gastrulation stage (HH4) to day 4 of development, its expression in the developing inner organs and limbs, and we compare its expression to the expression of Ask1 during mouse development. Furthermore, we provide evidence that FGF signaling is required for ASK1 expression in chick nasal mesenchyme. In contrast, expression in the mouse nasal region was restricted to the epithelium and was independent of FGF signaling. Our analysis demonstrates that ASK1 has a spatially restricted and temporally dynamic expression pattern in both chick and mouse embryos, which includes conserved as well as species-specific expression domains.

Apoptosis in liver disease

The description of the morphological hallmarks of programmed cell death, apoptosis, in 1972 by Kerr, Wyllie and Currie started a field of research that revolutionized our understanding of cellular proliferation, tissue homeostasis and pathophysiology of many diseases. In the following years, a series of proteins involved in signaling and intracellular death pathways were identified and 30 years later the Noble Prize for physiology and medicine was awarded to S. Brenner, H. R. Horvitz and J. E. Sulston for their discoveries related to describing the mechanisms of cell death (apoptosis). The delineation of the signaling pathways that mediate apoptosis changed the paradigms of understanding in many liver diseases. The most detailed analyzed mode of apoptosis involves a cell surface-based receptor-ligand system. Death receptors are typically members of the tumor necrosis factor-receptor superfamily and comprise an intracellular death domain. Following ligand binding to the receptor, intracellular adapter molecules are recruited to the receptor and subsequently transmit the apoptotic signal. Intracellular organelle-dependent signaling occurs, and effector molecules then augment the receptor-initiated apoptosis process. Cell death and degradation follows the activation of a highly regulated set of cytosolic and nuclear proteases and DNAses. Receptor-independent activation of the apoptotic process can occur as part of the cytotoxicity related to UV radiation, chemotherapeuticals or other DNA-damaging agents through activation of intracellular sensors of cellular integrity, e.g. the tumor suppressor gene p53. In contrast to necrosis, apoptosis is not commonly accompanied by an inflammatory response that causes collateral cell damage. The apoptotic program is highly effective in eliciting cell death and thus must be tightly controlled. This is achieved through continuous integration of pro- and antiapoptotic signals at the individual cell level. Dysregulation of the apoptotic process, resulting in too much or too little cell death, has potentially devastating effects and has been implicated in many forms of liver disease like acute liver failure or hepatocellular carcinoma. This review will focus initially on recent progress in signaling events of hepatocellular apoptosis and subsequently discuss the consequences for the hepatic pathophysiology that involves disarrangement of hepatocellular apoptosis.

Cyclic exposure to hypoxia and reoxygenation selects for tumor cells with defects in mitochondrial apoptotic pathways.

The negative influence of hypoxia on the outcome of malignant tumors may be caused by direct oxygen effects, and potentially, the selection of resistant tumor cells under repetitive hypoxia. To evaluate whether cyclic hypoxia selects for resistant cells and to analyze the underlying mechanisms, the influence of cyclic hypoxia on intracellular death pathways was determined in tumor cells. It could be demonstrated that cyclic hypoxia selects for cells with increased resistance against hypoxia-induced apoptosis. These cells exhibited a cross-resistance against paradigmatic triggers of mitochondrial apoptotic pathways (ionizing radiation/etoposide). In contrast, TRAIL-receptor mediated apoptosis remained unaffected. Thus, cyclic hypoxia selects for cells with defects of the mitochondrial rather than receptor-mediated pathways. Selection of p53-defective cells has been described as a consequence of cyclic hypoxia; therefore, we evaluated the impact of hypoxic selection on activation of p21 and downstream mediators of p53-dependent apoptosis. p53 function and protein levels of key mediators of mitochondrial apoptosis remained unaffected by hypoxic selection. However, radiation-induced conformational changes of Bax were reduced after cyclic hypoxia. In summary, it could be demonstrated that hypoxic stress confers a selection pressure on mitochondrial apoptotic pathways and, consecutively, to an increased resistance toward mitochondrial death triggers.

Targeting apoptosis in acute tubular injury

Recent research has shown that apoptosis and its regulatory mechanisms contribute to cell number regulation in acute renal failure. Acute tubular necrosis is the most frequent form of parenchymal acute renal failure. The main causes are ischemia–reperfusion, sepsis and nephrotoxic drugs. Exogenous factors such as nephrotoxic drugs and bacterial products, and endogenous factors such as lethal cytokines promote tubular cell apoptosis. Such diverse stimuli engage intracellular death pathways that in some cases are stimulus-specific. We now review the role of apoptosis in acute renal failure, the potential molecular targets of therapeutic intervention, the therapeutic weapons to modulate the activity of these targets and the few examples of therapeutic intervention on apoptosis.

Review : Apoptosis and Spinal Cord Injury

Apoptosis is the morphological counterpart of active, genetically programmed cell death and is important in development, immune function, and carcinogenesis. Recent data suggest that apoptosis may be important in neurodegenerative disorders, ischemic brain injury, and neurotrauma as well. Here we review very recent data from our laboratory and others that show that at least some of the pronounced secondary injury that follows spinal cord injury (SCI) may be caused by apoptosis and associated intracellular death pathways. Both neurons and glia seem to die by apoptosis; the response of oligodendrocytes in long tracts undergoing Wallerian degeneration is particularly long lived and may be responsible for chronic demyelination and some of the dysfunction in chronic SCI. These findings suggest that the therapeutic window for treatment of acute SCI may extend into the chronic phase. In addition, proliferation of ependymal cells occurs in concert with cell death, suggesting that both degeneration and repair may occur at the same time. Therapies aimed at altering the balance between these cellular events may be useful for future treatments of SCI. NEURO SCIENTIST 4:163-171, 1998