Caspase Inhibitor Research Articles

Coronaviruses exploit a host cysteine-aspartic protease for replication.

Highly pathogenic coronaviruses, including severe acute respiratory syndrome coronavirus 2(refs.1,2) (SARS-CoV-2), Middle East respiratory syndrome coronavirus3 (MERS-CoV) and SARS-CoV-1(ref.4), vary in their transmissibility and pathogenicity. However, infection by all three viruses results in substantial apoptosis in cell culture5-7 and in patient tissues8-10, suggesting a potential link between apoptosis and pathogenesis of coronaviruses. Here we show that caspase-6, a cysteine-aspartic protease of the apoptosis cascade, serves as an important host factor for efficient coronavirus replication. We demonstrate that caspase-6 cleaves coronavirus nucleocapsid proteins, generating fragments that serve as interferon antagonists, thus facilitating virus replication. Inhibition of caspase-6 substantially attenuates lung pathology and body weight loss in golden Syrian hamsters infected with SARS-CoV-2 and improves the survival of mice expressing human DPP4 that are infected with mouse-adapted MERS-CoV. Our study reveals how coronaviruses exploit a component of the host apoptosis cascade to facilitate virus replication.

Schweinfurthin induces ICD without ER stress and caspase activation

ABSTRACT Our previous study showed that one of the schweinfurthin compounds, 5’-methoxyschweinfurthin G (MeSG), not only enhances the anti-tumor effect of anti-PD1 antibody in the B16F10 murine melanoma model, but also provokes durable, protective anti-tumor immunity. Here we further investigated the mechanisms by which MeSG treatment induces immunogenic cell death (ICD). MeSG induced significant cell surface calreticulin (CRT) exposure in a time and concentration dependent manner as well as increased phagocytosis of tumor cells by dendritic cells in vitro. Interestingly, this CRT exposure differs from the canonical pathway in several aspects. MeSG does not cause ER stress and does not require PERK to induce CRT exposure. Caspase inhibitors partially rescue cells from MeSG-induced apoptosis, but fail to reduce CRT exposure. MeSG does not cause ERp57 exposure and the absence of ERp57 expression does not reduce CRT exposure. Finally, an intact ER to Golgi transport system is required for this phenomenon. These results lend support to the development of the schweinfurthin family as drugs to enhance clinical response to immunotherapy and highlight the need for additional research on the mechanisms of ICD induction.

Caspase Activation and Inhibition.

Caspase Activation and Inhibition.

Development of a bioluminescence assay for BIR2- caspase3 interaction through split luciferase complementary assay

Harnessing of firefly luciferase biochemistry enabled to develop bioluminescent assay for different intracellular protein-protein interactions. Caspase-3 and XIAP are two important players in maintaining the balance of cell death and survival. Linker-BIR2 from XIAP is known to be sufficient for inhibition of caspase-3. Here, caspase-3 and Linker-BIR2 were used for designing two reporters based on split luciferase complementary assay to develop a luminescent probe. Caspase3-CLuc and BIR2-NLuc fragments were ligated in pET-28a (+), overexpressed in E. coli BL21 (DE3) and purified to homogeneity. Interaction between caspase-3 and BIR2 was evaluated by different experimental methods such as luciferase and caspase-3 activity, directed mutagenesis and ELISA, which approved their specific interaction. Next, the developed reporters were employed for analyzing four compounds (Sulphadimidin, Glyburide, Carboplatin and Cetirizine) that showed effects on caspase-3 and BIR2 interaction with the largest effect for Carboplatin. Molecular docking was also showed the largest binding affinity for Glyburide. In conclusion, the developed assay showed specific interaction with light generation, and ability to analyze some chemicals in vitro.

Indication of the impact of environmental stress on the responses of the bivalve mollusk Unio tumidus to ibuprofen and microplastics based on biomarkers of reductive stress and apoptosis

The vulnerability of bivalve mollusks to micropollutants is estimated mainly in single model exposures. However, chronic environmental stress and complex exposures can modulate their responses. To evaluate the impact of population-dependent adaptations on the ability to react to common micropollutants, we compared freshwater bivalves Unio tumidus from two distinct populations, pure (Pr) and contaminated (Ct), in their exposures to microplastics (MP, 1 mg L−1, size 0.1–0.5 mm), pharmaceutical ibuprofen (IBU, 0.8 μg L−1), or their combination (Mix) for 14 days. Control groups from both sites showed remarkable differences, with lower levels of total antioxidant capacity (TAC), metallothionein protein (MTSH), NADH and NAD+, cytochrome P450-related EROD, glutathione-S transferase (GST), and citrate synthase (CS) but higher levels of GSH, GSSG, caspase-3 and cathepsin D (CTD) in the Ct-control group. These data indicate a chronic stress impact in the Ct population. Under exposures, we found an almost common strategy in both populations for NAD+/NADH and MTSH suppression and CTD induction. Additionally, Mix exposure caused an increase in CS, and IBU did not change GSH in both populations. However, the expected response to IBU – the suppression of caspase-3 – was indicated only in PrIBU- and PrMix-mollusks. CTD efflux increased dramatically only in PrMP- and PrMix- groups, and suppression of EROD and GST was detected in the PrMix-group. According to discriminant analysis, exposed Pr-groups were highly differentiated from control, whereas Ct-control and exposed groups had common localization demonstrating high resistance to environmental stress. Thus, the same exposures resulted in different adverse outcome pathways depending on the population.

Sanguinarine Induces H2O2-Dependent Apoptosis and Ferroptosis in Human Cervical Cancer.

Sanguinarine (SNG) is a benzophenanthridine alkaloid isolated mainly from Sanguinaria canadensis, Chelidonium majus, and Macleaya cordata. SNG is considered an antineoplastic agent based on its cytotoxic activity against various tumors. However, the exact molecular mechanism through which SNG mediates this activity has not been elucidated. Here, we report that SNG induces death in human cervical cancer (HeLa) cells through activation of two interdependent cell death pathways—apoptosis and ferroptosis. SNG-induced apoptosis was characterized by caspase activation and PARP cleavage, while ferroptosis involved solute carrier family 7 member 11 (SLC7A11) down-regulation, glutathione (GSH) depletion, iron accumulation, and lipid peroxidation (LPO). Interestingly, incubation with caspase inhibitor z-VAD-fmk not only inhibited the features of apoptosis, but also negated markers of SNG-induced ferroptosis. Similarly, pretreatment with ferroptosis inhibitor ferrostatin-1 (Fer-1), apart from rescuing cells from SNG-induced ferroptosis, also curbed the features of SNG-induced apoptosis. Our study implies that, together, apoptosis and ferroptosis act as partners in the context of SNG mediated tumor suppression in HeLa cells. Importantly, SNG increased the generation of reactive oxygen species (ROS), and ROS inhibition blocks the induction of both apoptosis and ferroptosis. These findings highlight the value of continued investigation into the potential use of SNG as an antineoplastic agent.

ABT-263, an Inhibitor of Bcl-2 Family Antiapoptotic Proteins, Sensitizes Prometaphase-Arrested HeLa Cells to Apoptosis Induced by Mild Hyperthermia

Aims: We have previously shown that mild hyperthermia (39-42°C) rapidly induces apoptosis in H-HeLa cells arrested in mitosis but not in interphase cells and not in mitotic-arrested cells of other HeLa strains such as HeLa S3. Our goal was to test the idea that this sensitivity to hyperthermia is due to down-regulation of Bcl-2 family antiapoptotic proteins during mitosis.
 Methodology: HeLa S3 cells were arrested in mitosis with spindle poisons and simultaneously treated with mild hyperthermia (42°C) and ABT-263 (Navitoclax), an inhibitor of Bcl-2 and other anti-apoptotic proteins. Apoptosis was observed microscopically and confirmed by observation of procaspase 3 cleavage using western immunoblotting.
 Results: Treatment of prometaphase-arrested HeLa S3 cells with mild hyperthermia (42°C) and as little as 200 nM ABT-263 induces apoptosis in the great majority of cells within 2 hr, as judged by blebbing of plasma membranes. The effect is not seen with interphase cells and is blocked by caspase inhibitors z-VAD-fmk and z-DEVD-fmk.
 Conclusion: Our results show that inhibition of Bcl-2 family antiapoptotic proteins sensitizes prometaphase-arrested HeLa cells, but not interphase cells, to apoptosis induced by mild hyperthermia. An open question is why are prometaphase-arrested cells more susceptible than interphase cells? If the features of mitotic-arrested cells that make them sensitive could be identified and mimicked in interphase cells by appropriate drug treatments, it may be possible to combine systemic chemotherapy with local hyperthermia to provide an effective new treatment for tumors.

The Anti-Apoptotic Effects of Caspase Inhibitors in Propyl Gallate-Treated Lung Cancer Cells Are Related to Changes in Reactive Oxygen Species and Glutathione Levels.

Propyl gallate [3,4,5-trihydroxybenzoic acid propyl ester; PG] exhibits an anti-growth effect in various cells. In this study, the anti-apoptotic effects of various caspase inhibitors were evaluated in PG-treated Calu-6 and A549 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. Treatment with 800 μM PG inhibited the proliferation and induced the cell death of both Calu-6 and A549 cells at 24 h. Each inhibitor of pan-caspase, caspase-3, caspase-8, and caspase-9 reduced the number of dead and sub-G1 cells in both PG-treated cells at 24 h. PG increased ROS levels, including O2∙−, in both lung cancer cell lines at 24 h. Generally, caspase inhibitors appeared to decrease ROS levels in PG-treated lung cancer cells at 24 h and somewhat reduced O2∙− levels. PG augmented the number of GSH-depleted Calu-6 and A549 cells at 24 h. Caspase inhibitors did not affect the level of GSH depletion in PG-treated A549 cells but differently and partially altered the depletion level in PG-treated Calu-6 cells. In conclusion, PG exhibits an anti-proliferative effect in Calu-6 and A549 lung cancer cells and induced their cell death. PG-induced lung cancer death was accompanied by increases in ROS levels and GSH depletion. Therefore, the anti-apoptotic effects of caspase inhibitors were, at least in part, related to changes in ROS and GSH levels.

Apoptotic caspases suppress an MDA5-driven IFN response during productive replication of human papillomavirus type 31

Human papillomaviruses (HPVs) infect the basal proliferating cells of the stratified epithelium, but the productive phase of the life cycle (consisting of viral genome amplification, late gene expression, and virion assembly) is restricted to the highly differentiated suprabasal cells. While much is known regarding the mechanisms that HPVs use to block activation of an innate immune response in undifferentiated cells, little is known concerning how HPV prevents an interferon (IFN) response upon differentiation. Here, we demonstrate that high-risk HPVs hijack a natural function of apoptotic caspases to suppress an IFN response in differentiating epithelial cells. We show that caspase inhibition results in the secretion of type I and type III IFNs that can act in a paracrine manner to induce expression of interferon-stimulated genes (ISGs) and block productive replication of HPV31. Importantly, we demonstrate that the expression of IFNs is triggered by the melanoma differentiation-associated gene 5 (MDA5)-mitochondrial antiviral-signaling protein (MAVS)-TBK1 (TANK-binding kinase 1) pathway, signifying a response to double-stranded RNA (dsRNA). Additionally, we identify a role for MDA5 and MAVS in restricting productive viral replication during the normal HPV life cycle. This study identifies a mechanism by which HPV reprograms the cellular environment of differentiating cells through caspase activation, co-opting a nondeath function of proteins normally involved in apoptosis to block antiviral signaling and promote viral replication.

Biological Effects and Mechanisms of Caspases in Early Brain Injury after Subarachnoid Hemorrhage

Caspases are an evolutionarily conserved family of proteases responsible for mediating and initiating cell death signals. In the past, the dysregulated activation of caspases was reported to play diverse but equally essential roles in neurodegenerative diseases, such as brain injury and neuroinflammatory diseases. A subarachnoid hemorrhage (SAH) is a traumatic event that is either immediately lethal or induces a high risk of stroke and neurological deficits. Currently, the prognosis of SAH after treatment is not ideal. Early brain injury (EBI) is considered one of the main factors contributing to the poor prognosis of SAH. The mechanisms of EBI are complex and associated with oxidative stress, neuroinflammation, blood-brain barrier disruption, and cell death. Based on mounting evidence, caspases are involved in neuronal apoptosis or death, endothelial cell apoptosis, and increased inflammatory cytokine-induced by apoptosis, pyroptosis, and necroptosis in the initial stages after SAH. Caspases can simultaneously mediate multiple death modes and regulate each other. Caspase inhibitors (including XIAP, VX-765, and Z-VAD-FMK) play an essential role in ameliorating EBI after SAH. In this review, we explore the related pathways mediated by caspases and their reciprocal regulation patterns after SAH. Furthermore, we focus on the extensive crosstalk of caspases as a potential area of research on therapeutic strategies for treating EBI after SAH.

Commentary on “PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons”

Commentary on “PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons”

Pharmacological and Genetic Inhibition of HDAC4 Alleviates Renal Injury and Fibrosis in Mice.

Histone deacetylase 4 (HDAC4) has been shown to be involved in cell proliferation, differentiation, and migration and is associated with a variety of cancers. However, the role of HDAC4 in renal fibrogenesis and its mechanisms are unclear. We assessed the role of HDAC4 and possible mechanisms of fibrosis in a murine model of kidney injury induced by unilateral ureteral obstruction (UUO) using tasquinimod, a highly selective HDAC4 inhibitor, and knockout mice with depletion of HDAC4 in renal tubular cells. UUO injury resulted in increased expression of HDAC4 and fibrotic proteins fibronectin and α-smooth muscle actin, while treatment with tasquinimod or knockout of HDAC4 significantly reduced their expression. Pharmacological and genetic inhibition of HDAC4 also decreased tubular epithelial cell arrest in the G2/M phase of the cell cycle, expression of transforming growth factor-β1 and phosphorylation of Smad3, signal transducer and activator of transcription 3, and extracellular signal-regulated kinase 1/2 in the injured kidney. Moreover, tasquinimod treatment or HDAC4 deletion inhibited UUO-induced renal tubular cell injury and apoptosis as indicated by reduced expression of neutrophil gelatinase–associated lipocalin, Bax, and inhibition of caspase-3. Finally, administration of tasquinimod or knockdown of HDAC4 prevented injury-related repression of Klotho, a renoprotective protein. Our results indicate that HDAC4 is critically involved in renal tubular injury and fibrosis and suggest that HDAC4 is a potential therapeutic target for treatment of chronic fibrotic kidney disease.

Decreased S Phase and G2/M Phase Cells by a Bioactive Fraction from the Hexane Extract of Rhododendron aroboreum Leading to the Apoptosis of HeLa Cells In-Vitro

Search for novel anticancer compounds from natural sources is a priority goal of cancer biologists. Rhododendron arboreum is one of the most stately and impressive Rhododendron species. In hilly areas, the flowers of R. arboreum are used for the treatment of chronic diseases. The current study was aimed to evaluate the anti-proliferative property of R. arboreum on HeLa cells. Crude extract of the flower was prepared using serial extraction procedure with different solvents. Cytotoxicity of the extracts on HeLa, CHO and human lymphocytes were assessed using MTT assay and trypan blue dye exclusion methods. The extract was further purified by TLC and characterized by LC-MS method. The hexane extract showed significant cytotoxicity to HeLa cells. This extract was fractionated using thin layer chromatography, 6 fractions were isolated, out of which fraction 3 showed the maximum cytotoxic activity with an IC50 of 15 μg/mL. The mechanism of action was observed as apoptosis induction as indicated by elevated caspase activity and inhibition of cell cycle with reduced cell population in the S and G2/M phases. LC-MS analysis of this promising bioactive fraction resulted in a molecular ion fragment with an m/z value of 437.48 which is corresponding to that of brefeldin A, an anticancer compound. It can be concluded that R. arboreum flower has cytotoxic activity which could be due to the presence of brefeldin A, that warrants further studies on purification and characterization towards the development of highly efficient therapeutics for cancer.
 HIGHLIGHTS
 
 Anti-cancer drug discovery from natural sources like medicinal herbs having diverse metabolites of therapeutic significance provide lead molecules towards cancer drug development
 The anti-proliferative/apoptotic potential of a bioactive fraction from Rhododendron aroboreum, was analysed on cancer cell lines by MTT, Lactate dehydrogenase, Caspase and cell cycle studies. The bioactive compound was characterized through preparative TLC and LC-MS methods
 The potent apoptotic potential of Rhododendron aroboreum flower extract was demonstrated through reduced S-phase and G2/M phase cells, increased caspase, LDH activities in our study results
 The presence of Brefeldin A, previously reported as an anticancer compound in a microbe, was observed for the 1st time in Rhododendron aroboreum through our study in LC-MS analysis
 
 GRAPHICAL ABSTRACT

Cordycepin enhances hyperthermia-induced apoptosis and cell cycle arrest by modulating the MAPK pathway in human lymphoma U937 cells.

Hyperthermia induces cancer cell death. However, the cytotoxic effect of hyperthermia is not sufficient. Cordycepin can also induce apoptosis in cancer cells and enhance the antitumoral activity of irradiation. To examine cordycepin-mediated enhancement of hyperthermia-induced apoptosis, this study investigated the combined effects and apoptotic mechanisms of hyperthermia and cordycepin on human leukemia U937 cells. Cell viability and apoptosis were measured using MTT assays, Hoechst 33342 staining and Annexin V/PI double staining. The distribution of the cell cycle and sub-G1 phase, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were examined by flow cytometry. The expression of related proteins was analyzed by western blotting. Combined treatment with hyperthermia and cordycepin markedly augmented apoptosis by upregulating Bax and suppressing Bcl-2, Bid and activated caspase 3 and 8 expression, and apoptosis was decreased by Z-VAD-fmk (a pan caspase inhibitor). We also found that the MMP was significantly decreased and excessive ROS generation occurred. The combination treatment also induced arrest in the G2/M phase by downregulating cyclin dependent kinase 1 (CDK1) and cyclin B1 protein expression. Furthermore, it was observed that mitogen-activated protein kinase (MAPK) pathway including ERK, JNK and p38 signals was involved in the induction of apoptosis. The phosphorylated p38 and JNK were increased and ERK phosphorylation was decreased by the combined treatment. In addition, N-acetyl-L-cysteine (NAC) significantly protected the cells by restoring ROS levels and the activity of caspase-3, inactivating the MAPK pathway. Cordycepin significantly enhanced hyperthermia-induced apoptosis and G2/M phase arrest in U937 cells. The combined treatment enhanced apoptosis through the MAPK pathway and mitochondrial dysfunction, and these effects could be rescued by NAC. We report for the first time that cordycepin can be used as a hyperthermia sensitizer to treat leukemia.

HDAC inhibitor and proteasome inhibitor induce cleavage and exosome-mediated secretion of HSP90 in mouse pluripotent stem cells

Heat shock protein 90 (HSP90), one of the molecular chaperones, stabilizes several proteins necessary to maintain pluripotency of embryonic stem (ES) cells. Recently, we reported that HDAC inhibitors and proteasome inhibitors down-regulate HSP90 activity through HSP90 cleavage induced by reactive oxygen species (ROS) generation and caspase 10 activation in various cancer cells. In this study, we investigated HSP90 cleavage in mouse ES cells. HDAC inhibitors and proteasome inhibitors induced HSP90 cleavage in the mouse ES cell line R1, and the cleaved HSP90 was barely found in the cells and instead secreted out of the cells through the exosome. The HSP90 cleavage was associated with ROS generation and caspase 10 activation. In addition, HDAC inhibitor and proteasome inhibitor induced Fas expression, and the inhibition of caspase 8, a downstream molecule of Fas, blocked HSP90 cleavage. Therefore, HDAC inhibitor- and proteasome inhibitor-mediated HSP90 cleavage was induced by ROS generation and Fas expression. We observed similar results in mouse induced pluripotent stem (iPS) cells. Taken together, HSP90 cleavage was induced in mouse pluripotent cells similarly to cancer cells but differently regulated through Fas expression and exosomal secretion. These findings will be helpful in elucidating the regulation of HSP90 upon stress in pluripotent stem cells.

Isobavachalcone induces cell death through multiple pathways in human breast cancer MCF-7 cells

To explore the effects of isobavachalcone (IBC) on cell death of human breast cancer MCF-7 cells and explore the possible mechanism. MCF-7 cells were treated with different concentrations of IBC, and the changes in cell proliferation were assessed using MTT assay. Apoptosis of MCF-7 cells following treatment with 10, 20, and 40 μmol/L IBC was analyzed using flow cytometry with annexin V-FITC/PI double staining and fluorescence microscopy, and the expressions of apoptosis- and autophagy-related proteins (Bax, Bcl-2, Akt, p-Akt, p62, and LC3) were detected with Western blotting. Electron microscopy was used to observe the changes in submicrostructure of the cells following treatment with 40 μmol/L IBC. JC-1 assay kit, ATP assay kit, and reactive oxygen species (ROS) kit were used to determine the effect of IBC on mitochondrial function of the cells. MTT assay showed that IBC significantly inhibited the proliferation of MCF-7 cells in a concentration- and time-dependent manner, with IC50 values of 38.46, 31.31, and 28.26 μmol/L at 24, 48, and 72 h, respectively. IBC also concentration-dependently induced apoptosis of MCF-7 cells. IBC-induced cell death was inhibited by z-VAD-fmk, a caspase inhibitor (P < 0.05), but not by the necroptosis inhibitor necrostatin-1 (Nec-1). Western blotting showed that IBC-induced MCF-7 cell apoptosis by increasing Bax expression and down-regulating the expressions of Bcl-2, Akt and p-Akt-473 (all P < 0.05). With the increase of IBC concentration, the expression of autophagy-related protein p62 and the LC3-II/I ratio increased progressively. Electron microscopy revealed the presence of autophagic bodies in IBC-treated MCF-7 cells. IBC treatment also resulted in decreased mitochondrial membrane potential and intracellular ATP level and increased ROS accumulation in MCF-7 cells (P < 0.05). IBC is capable of inducing both apoptosis and autophagy in MCF-7 cells, suggesting the potential value of IBC as a lead compound in the development of anti-breast cancer agents.

The effects of dragon fruit (Hylocereus polyrhizus) extract on indomethacin-induced stomach ulcer in rats.

The aim of this study was to investigate the effects of red dragon fruit (Hylocereus polyrhizus) extract (DFE) on the stomach in ulcer model induced by indomethacin in rats. Effects of DFE were evaluated in indomethacin-induced gastric damage model on Sprague-Dawley rats. Experimental model: all rats were fasted for 24 h. At the end of this period, DFE was administered to the ulcer-induced groups. One hour after this application, a dose of 25 mg/kg of indomethacin was applied by oral gavage to all groups except the HEALTHY and DFE1000 groups. Six hours after indomethacin administration, the rats were euthanized with high-dose anesthesia and the experiment was terminated. Macroscopic and microscopic analyses for investigating ulcerative area, molecular and biochemical analyses for oxidative damages investigation and molecular analyses for the effect mechanism of indomethacin and DFE were conducted on stomach tissues in the study. While oxidative stress-associated markers such as MDA, BAX, and Caspase 3 increased dramatically in the indomethacin group, GSH antioxidant levels decreased. It was observed that these parameters were significantly improved in DFE 500 mg/kg and DFE 1000 mg/kg groups compared to ulcer group, and the results of especially DFE 1000 mg/kg group were similar to famotidine group. We observed that our histopathological findings also supported all our other findings. Dragon fruit extract was protected against indomethacin-induced ulcer damage by decreased MDA levels, increased GSH levels, and inhibition of Caspase 3, BAX, and Cox-2, and activation of Cox-1. PRACTICAL APPLICATIONS: People of all ages around the world suffer from gastric ulcer which is one of the most common gastrointestinal ailments. The etiological factors of the disease are using of cigarette and alcohol, nutritional deficiencies, infections, and using non-steroidal anti-inflammatory drugs which use frequent and indiscriminate. Indomethacin is one of the NSAIDs and is commonly preferred to induce ulcer modeling in rats due to its gastric toxicity potential. Current anti-ulcer drugs have many serious side effects. Patients who suffered from gastric ulcer tend to discontinue the drug because of side effects. Therefore, patients need new agents that are non-toxic, have few side effects, and are easily accessible anti-ulcer drugs. Dragon fruit, as a medicinal herb, is highly valuable and widely used in traditional medicine, and may provide gastroprotective activity. Studies have shown that H. polyrhizus has antioxidant activities. We consider the effects of dragon fruit extract (DFE) to be a therapeutic drug for an indomethacin-induced ulcer model.

Abstract 2998: Inhibition of MDM2-p53 interaction by alrizomadlin (APG-115) induces pyroptotic cell death in gasdermin E (GSDME)-expressing cancer cells

Abstract Mouse double minute-2 (MDM2)-p53 inhibitor alrizomadlin (APG-115) is an investigational agent known to induce apoptosis of TP53-wild type cancer cells (Aguilar et al, J Med Chem 2017). Emerging evidence suggests that activation of p53 by alrizomadlin also promotes antitumor immunity in the tumor microenvironment (Fang et al, JITC 2019; Zhou et al, Nat Immunol 2021), but the links between these processes are incompletely understood. Pyroptosis refers to inflammatory programmed cell death. Central to this process is the family of gasdermins, which can form pores in cell plasma membranes, resulting in lysis and release of immune stimulants. In cells expressing these proteins, GSDME can be cleaved by caspase-3, which converts noninflammatory apoptosis to pyroptosis (Zhang et al, Nature 2020). In this context, caspase-3/GSDME appears to represent a switch between apoptosis and pyroptosis. Given that alrizomadlin elicits its apoptogenic activity primarily by activating caspase-3, we hypothesized that the MDM2-p53 inhibitor might also induce pyroptosis in GSDME-expressing cells by cleaving caspases. Our study demonstrated that alrizomadlin uniformly elicited robust pyroptosis in GSDME-expressing cancer cells. After treatment with alrizomadlin, the dying cells showed swelling with plasma membrane blebbing characteristic of pyroptosis. Alrizomadlin treatment upregulated biomarkers of pyroptosis, inducing activation of caspase-3, promoting cleavage of GSDME, and increasing release of lactate dehydrogenase (LDH). After a short drug exposure of 24 hours, alrizomadlin elicited concurrent apoptotic and pyroptotic tumor cell death, as evidenced by coexistence of the hallmarks of apoptosis (PARP-1 cleavage) and pyroptosis (caspase-3 cleavage and activated GSDME) markers. When the treatment duration was extended to 48 hours, cleavage of GSDME continued to increase whereas PARP-1 cleavage became nearly undetectable, suggesting a conversion from apoptosis to pyroptosis. Inhibition of caspases by pan-caspase inhibitor Q-VD-OPh (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone) abolished not only alrizomadlin-induced caspase-3 and GSDME cleavage but also reduced LDH release and cell death without inducing morphologic changes associated with pyroptosis. Taken together, these results suggest that, in addition to apoptosis, MDM2-p53 inhibitor alrizomadlin induces caspase-mediated pyroptosis in GSDME-expressing cancer cells. In this study, we reveal for the first time that apoptosis-inducing, alrizomadlin induces both apoptosis and pyroptosis in GSDME-expressing cancer cells. GSDME-dependent pyroptosis may be a previously unrecognized mechanism of action for alrizomadlin to exert antitumor immunity, with potentially important implications for clinical development of therapy involving MDM2-p53 inhibition. Citation Format: Qiuqiong Tang, Douglas D. Fang, Huidan Yu, Bingxing Wu, Yan Yin, Dajun Yang, Yifan Zhai. Inhibition of MDM2-p53 interaction by alrizomadlin (APG-115) induces pyroptotic cell death in gasdermin E (GSDME)-expressing cancer cells [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 2998.

Abstract 2605: BH3 mimetics synergize with the Wee1 inhibitor ZN-c3 by activating caspases which induce DNA damage and degrade key proteins

Abstract Background: Wee1 is a crucial cell cycle checkpoint kinase that regulates the G2/M checkpoint in response to DNA damage, and its inhibition can cause mitotic catastrophe and apoptosis in tumor cells. Inhibitors of BCL-2 have been shown to also induce apoptosis. ZN-d5 and ZN-c3, currently in clinical development for the treatment of cancer, are highly selective and potent inhibitors of Bcl-2 and Wee1, respectively. The combination of ZN-d5 with ZN-c3 induced robust tumor regressions in preclinical models and the mechanism of action of this activity is presented. Methods: Cell proliferation in tumor cell lines was measured using CellTiter-Glo® (Promega). Cleaved caspase-3, cleaved PARP, phosphorylated CDK1 (p-CDK1), γH2AX, Wee1, RRM2 and other markers were measured by Western blotting, IHC and/or flow cytometry. Anti-tumor efficacy was determined in several xenograft models. Results: The combination of ZN-c3 and ZN-d5 resulted in additive or synergistic anti-proliferative activity in several tumor cell lines. This enhancement of anti-proliferative activity was also observed at concentrations of ZN-d5 that induced caspase activation, but minimal apoptosis. Treatment of tumor cell lines with ZN-d5 alone at these sublethal concentrations caused induction of γH2AX, a marker of double-stranded DNA breaks, and degradation of caspase-sensitive proteins relevant to the DNA Damage Response (DDR), such as RRM2 (a ribonucleotide reductase subunit) and Wee1. Degradation of Wee1 correlated with decreases in p-CDK1, a marker of Wee1 inhibition. These effects were mainly reversible and were inhibited by pre-treatment with caspase inhibitors. Mechanistically, the combination of ZN-c3 and ZN-d5 resulted in increased levels of γH2AX, enhanced inhibition of p-CDK1, and increased levels of apoptotic markers compared to single agent treatment. Similar results were obtained when ZN-c3 was combined with inhibitors of other BCL-2 family proteins such as inhibitors of MCL-1 and BCL-xL. Finally, the combination of ZN-c3 with ZN-d5 or other BH3 mimetics showed synergistic anti-tumor activity in several xenograft models further supporting the rationale for combining these agents. Conclusions: ZN-d5 or other BH3 mimetics caused caspase-mediated DNA damage and degraded relevant proteins such as Wee1 and RRM2 resulting in additive or synergistic anti-tumor activity when combined with ZN-c3. These results support a novel use of BH3 mimetics to activate caspase activity, independently of apoptosis induction, to significantly enhance the activity of DDR inhibitors such as ZN-c3 Citation Format: Hooman Izadi, Noah Ibrahim, Tiffany Hoang, Jianhui Ma, Petrus R. de Jong, Joseph Pinchman, Kevin D. Bunker, Ahmed A. Samatar, Fernando Doñate. BH3 mimetics synergize with the Wee1 inhibitor ZN-c3 by activating caspases which induce DNA damage and degrade key proteins [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 2605.

Abstract 3702: UBE4B interacts with the ITCH E3 ubiquitin ligase to induce Ku70 and c-FLIPL K63/K48 branched polyubiquitination and enhanced neuroblastoma HDACi-mediated apoptosis

Abstract Expression of the UBE4B ubiquitin ligase is associated with neuroblastoma patient outcomes, but the functional roles of UBE4B in neuroblastoma pathogenesis are not known. We evaluated interactions of UBE4B with the E3 ubiquitin ligase ITCH/AIP4 and the effects of UBE4B expression on Ku70, c-FLIPL, and p53 ubiquitination and proteasomal degradation by co-immunoprecipitation and Western blots. We also evaluated the role of UBE4B in apoptosis induced by HDAC inhibition in our neuroblastoma cell lines model using Western blots. UBE4B was found in a complex with ITCH, with binding mediated by WW domains in the ITCH protein. ITCH activation led to ITCH-UBE4B E3-E4 ubiquitin ligase complex formation and recruitment of Ku70 and c-FLIPL via ITCH WW domains, followed by Ku70 and c-FLIPL Lys48/Lys63 branched polyubiquitination and proteasomal degradation. Histone deacetylase (HDAC) inhibition induced Ku70 and c-FLIPL acetylation, leading to release of c-FLIPL and Bax from Ku70, increased Ku70 and c-FLIPL Lys48/Lys63 branched polyubiquitination via the ITCH-UBE4B complex, and induction of apoptosis. UBE4B depletion in our neuroblastoma cell lines model led to reduced polyubiquitination and increased levels of Ku70 and c-FLIPL proteins leading to the massive reduction of apoptosis induced by HDAC inhibition via stabilization of Ku70 and c-FLIPL proteins allowing the inhibition of the full caspase 8 activation. Our results have identified novel interactions and novel targets for UBE4B ubiquitin ligase activity and a direct role of the ITCH-UBE4B complex in responses of neuroblastoma cells to HDAC inhibition, suggesting that the ITCH-UBE4B complex plays a critical role in responses of neuroblastoma to therapy and suggesting a potential mechanism underlying the association of UBE4B expression with neuroblastoma patient outcomes. Citation Format: Christophe Le Clorennec, Divya Subramonian, Yuchen Huo, Peter E. Zage. UBE4B interacts with the ITCH E3 ubiquitin ligase to induce Ku70 and c-FLIPL K63/K48 branched polyubiquitination and enhanced neuroblastoma HDACi-mediated apoptosis [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 3702.