Caspase Inhibitor Research Articles

Caspase-8 inhibition improves the outcome of bacterial infections in mice by promoting neutrophil activation

During differentiation, neutrophils undergo a spontaneous pro-inflammatory program that is hypothesized here to be under caspase-8 control. In mice, intraperitoneal administration of the caspase-8 inhibitor z-IETD-fmk is sufficient to unleash the production of pro-inflammatory cytokines and neutrophil influx in the absence of cell death. These effects are due to selective inhibition of caspase-8 and require tonic interferon-β (IFN-β) production and RIPK3 but not MLKL, the essential downstream executioner of necroptotic cell death. Invitro, stimulation with z-IETD-fmk is sufficient to induce significant cytokine production in murine neutrophils but not in macrophages. Therapeutic administration of z-IETD-fmk improves clinical outcome in models of lethal bacterial peritonitis and pneumonia by augmenting cytokine release, neutrophil influx, and bacterial clearance. Moreover, the inhibitor protects mice against high-dose endotoxin shock. Collectively, our data unveil a RIPK3- and IFN-β-dependent pathway that is constitutively activated in neutrophils and can be harnessed therapeutically using caspase-8 inhibition.

Caspase-9 inhibition confers stronger neuronal and vascular protection compared to VEGF neutralization in a mouse model of retinal vein occlusion.

Retinal vein occlusion (RVO) is a sight-threatening condition typically treated with intravitreal injection of vascular endothelial growth factor (VEGF) antagonists. Treatment response to anti-VEGF therapies is highly variable, with poor visual outcomes and treatment response in patients with significant retinal nonperfusion following RVO. Recently, caspase-9 has been identified as a potent regulator of edema, gliosis, and neuronal dysfunction during acute retinal hypoxia. The purpose of this study was to compare the therapeutic effect of caspase-9 inhibition against VEGF-neutralization in an established mouse model of RVO. Adult male C57Bl/6 J mice were randomized to induction of RVO and treatment with either vehicle, intravitreal injection of anti-VEGF antibody, topical administration of a selective caspase-9 inhibitor (Pen1-XBir3), or a combination therapy. Animals were followed on days 1, 2, and 8 after RVO with fundus retinal imaging, and with optical coherence tomography (OCT) to capture retinal swelling, capillary nonperfusion (measured by disorganization of retinal inner layers, DRIL), hyperreflective foci (HRF), and retinal atrophy. Focal electroretinography (ERG) measurements were performed on day 7. Histology was performed on retinal sections from day 8. Both VEGF neutralization and caspase-9 inhibition showed significant retinal protection from RVO compared to vehicle treatment arm. Retinal reperfusion of occluded veins was accelerated in eyes receiving caspase-9 inhibitor, but not significantly different from vehicle in the anti-VEGF group. Retinal edema was suppressed in all treatment groups, with approximately 2-fold greater edema reduction with caspase-9 inhibition compared to VEGF neutralization. HRF were reduced similarly across all treatment groups compared to vehicle. Retinal detachment was reduced only in eyes treated with caspase-9 inhibitor monotherapy. Caspase-9 inhibition reduced retinal atrophy and preserved ERG response; VEGF neutralization did not prevent neurodegeneration following RVO. Caspase-9 inhibition confers stronger neuronal and vascular protection compared to VEGF neutralization in the mouse laser-induced model of RVO.

The Effect of MSCs with/without Silymarin Against Liver Fibrosis in HepG2 Cell Line with Focused on Caspase3, NFKβ, Apoptosis, Proliferation, Necrosis, and Collagen

Background: Liver fibrosis is characterized by irreversible damage to the liver parenchyma. The mechanism of fibrosis is based on the activation of collagen, nuclear factor kappa (NF-kβ) activation, and inhibition of ne-crosis, apoptosis, caspase-3 and proliferation. The HepG2 cell line is an evolved human liver cancer cell that exhibits a human hormone response. Hepatocyte stem cells have antifibrotic, anti-inflammatory, and im-munomodulatory properties, can suppress apoptosis and necrosis, and promote proliferation. Methods: This is an experimental in vitro study using pre-test and post-test. Hepatocyte stem cells were obtained from the neonatal umbilical cord. The cell lines were divided into four groups. Analysis was performed on below variable including caspase-3, NF-kβ, necrosis, apoptosis, proliferation and cytopathological capacity. Results: Cytopathological studies of the hepG2 cell line showed fibroblast growth. Mesenchymal stem cells reduced NF-kβ activity, but not caspase 3 activity. Mesenchymal stem cells also significantly reduced apoptosis and necrotic activity, but silymarin was not significantly affected. Mesenchymal stem cells also significantly increased proliferation (p <0.001), but silymarin was not significantly affected. Conclusion: Mesenchymal stem cells significantly inhibited NF-kβ, necrosis, apoptosis, and proliferation ac-tivation, but caspase-3 inhibition was not significantly affected.

1724-P: Caspase 3/7 Activation and RIPK3 Are Dispensable for Synthetic hIAPP-Induced ß-Cell Death

Islet amyloid is a hallmark of type 2 diabetes that is associated with β-cell loss. Aggregation of the β-cell secretory product human islet amyloid polypeptide (hIAPP) to form islet amyloid promotes β-cell death 1) via direct effects on the β cell and 2) by stimulating islet immune cell cytokine production. We recently showed that β cells are susceptible to TNFα-induced necroptosis, a form of caspase-independent, receptor interacting protein kinase 3 (RIPK3)-mediated cell death. We hypothesized that RIPK3 also promotes β-cell death in response to islet amyloid formation. To test this hypothesis, we evaluated INS-1, NIT-1 and isolated mouse islet cells treated with synthetic hIAPP (20 μM) +/− the pan-caspase inhibitor zVAD (40 μM). We found synthetic hIAPP increased cell death and caspase activity in INS-1, NIT-1 and mouse islet cells over 48 h. Remarkably, inhibition of caspases with zVAD (as measured by caspase 3/7 activity) failed to reduce synthetic hIAPP-mediated cell death in INS-1 (n=3, p=0.99), NIT-1 (n=3, p=0.99) or mouse islet cells (n=4, p=0.98). To evaluate RIPK3 in hIAPP-induced β-cell death, we examined a small molecule RIPK3 inhibitor (GSK’872, 5 μM), gene-edited NIT-1 Ripk3Δ cells (gRNA targeting exon 6), and intact isolated Ripk3−/− mouse islets. We found GSK’872 failed to protect INS-1 (n=3, p=0.32) or NIT-1 (n=3, p=0.30) cells from hIAPP-induced cell death over 48 h. Similarly, neither NIT-1 Ripk3Δ cells (n=2, hIAPP: p=0.82, hIAPP+zVAD: p=0.97) nor Ripk3−/− islets (n=4, hIAPP: p=0.63, hIAPP+zVAD: p=0.86) were protected from synthetic hIAPP-induced cell death either when caspases were active or inhibited. Our data indicate that caspase 3/7 activity and RIPK3 are dispensable for synthetic hIAPP-induced β-cell death in vitro. However, we found that RIPK3 is upregulated in response to endogenous islet amyloid formation in vitro. Future studies will determine whether RIPK3 promotes β-cell loss in part via effects on endogenous amyloid-induced cytokine signaling. Disclosure N.Mukherjee: None. C.J.Contreras: None. L.Lin: None. E.P.Cai: None. S.E.Kahn: Advisory Panel; Anji Pharmaceuticals, Bayer Inc., Boehringer Ingelheim Inc., Eli Lilly and Company, Merck & Co., Inc., Other Relationship; Novo Nordisk. A.T.Templin: None. Funding U.S. Department of Veterans Affairs (IK2BX004659 to A.T.T.); National Institutes of Health (T32DK064466 to C.J.C.); Indiana University School of Medicine (to N.M.)

Role of death-associated protein kinase 1 (DAPK1) in retinal degenerative diseases: an in-silico approach towards therapeutic intervention

The Death-associated protein kinase 1 (DAPK1) has emerged as a crucial player in the pathogenesis of degenerative diseases. As a serine/threonine kinase family member, DAPK1 regulates critical signaling pathways, such as apoptosis and autophagy. In this study, we comprehensively analyzed DAPK1 interactors and enriched molecular functions, biological processes, phenotypic expression, disease associations, and aging signatures to elucidate the molecular networks of DAPK1. Furthermore, we employed a structure-based virtual screening approach using the PubChem database, which enabled the identification of potential bioactive compounds capable of inhibiting DAPK1, including caspase inhibitors and synthetic analogs. Three selected compounds, CID24602687, CID8843795, and CID110869998, exhibited high docking affinity and selectivity towards DAPK1, which were further investigated using molecular dynamics simulations to understand their binding patterns. Our findings establish a connection between DAPK1 and retinal degenerative diseases and highlight the potential of these selected compounds for the development of novel therapeutic strategies. This study provides valuable insights into the molecular mechanisms underlying DAPK1-related diseases, and offers new opportunities for the discovery of effective treatments for retinal degeneration. Communicated by Ramaswamy H. Sarma

Carbon Quantum Dots from Roasted Coffee Beans: Their Degree and Mechanism of Cytotoxicity and Their Rapid Removal Using a Pulsed Electric Field.

Carbon quantum dots (CQDs) from heat-treated foods show toxicity, but the mechanisms of toxicity and removal of CQDs have not been elucidated. In this study, CQDs were purified from roasted coffee beans through a process of concentration, dialysis and lyophilization. The physical properties of CQDs, the degree and mechanism of toxicity and the removal method were studied. Our results showed that the size of CQDs roasted for 5 min, 10 min and 20 min were about 5.69 ± 1.10 nm, 2.44 ± 1.08 nm and 1.58 ± 0.48 nm, respectively. The rate of apoptosis increased with increasing roasting time and concentration of CQDs. The longer the roasting time of coffee beans, the greater the toxicity of CQDs. However, the caspase inhibitor Z-VAD-FMK was not able to inhibit CQDs-induced apoptosis. Moreover, CQDs affected the pH value of lysosomes, causing the accumulation of RIPK1 and RIPK3 in lysosomes. Treatment of coffee beans with a pulsed electric field (PEF) significantly reduced the yield of CQDs. This indicates that CQDs induced lysosomal-dependent cell death and increased the rate of cell death through necroptosis. PEF is an effective way to remove CQDs from roasted coffee beans.

Immunogenic cell death after combined treatment with radiation and ATR inhibitors is dually regulated by apoptotic caspases.

Inhibitors of the ATR kinase act as radiosensitizers through abrogating the G2 checkpoint and reducing DNA repair. Recent studies suggest that ATR inhibitors can also increase radiation-induced antitumor immunity, but the underlying immunomodulating mechanisms remain poorly understood. Moreover, it is poorly known how such immune effects relate to different death pathways such as caspase-dependent apoptosis. Here we address whether ATR inhibition in combination with irradiation may increase the presentation of hallmark factors of immunogenic cell death (ICD), and to what extent caspase activation regulates this response. Human lung cancer and osteosarcoma cell lines (SW900, H1975, H460, U2OS) were treated with X-rays and ATR inhibitors (VE822; AZD6738) in the absence and presence of a pan-caspase inhibitor. The ICD hallmarks HMGB1 release, ATP secretion and calreticulin surface-presentation were assessed by immunoblotting of growth medium, the CellTiter-Glo assay and an optimized live-cell flow cytometry assay, respectively. To obtain accurate measurement of small differences in the calreticulin signal by flow cytometry, we included normalization to a barcoded control sample. Extracellular release of HMGB1 was increased in all the cell lines at 72 hours after the combined treatment with radiation and ATR inhibitors, relative to mock treatment or cells treated with radiation alone. The HMGB1 release correlated largely - but not strictly - with loss of plasma membrane integrity, and was suppressed by addition of the caspase inhibitor. However, one cell line showed HMGB1 release despite caspase inhibition, and in this cell line caspase inhibition induced pMLKL, a marker for necroptosis. ATP secretion occurred already at 48 hours after the co-treatment and did clearly not correlate with loss of plasma membrane integrity. Addition of pan-caspase inhibition further increased the ATP secretion. Surface-presentation of calreticulin was increased at 24-72 hours after irradiation, but not further increased by either ATR or caspase inhibition. These results show that ATR inhibition can increase the presentation of two out of three ICD hallmark factors from irradiated human cancer cells. Moreover, caspase activation distinctly affects each of the hallmark factors, and therefore likely plays a dual role in tumor immunogenicity by promoting both immunostimulatory and -suppressive effects.

The natural chalcone cardamonin selectively induces apoptosis in human neuroblastoma cells

Neuroblastoma is the most common extracranial malignant tumor in childhood. Approximately 60% of all patients are classified as high-risk and require intensive treatment including non-selective chemotherapeutic agents leading to severe side effects. Recently, phytochemicals like the natural chalcone cardamonin (CD) have gained attention in cancer research. For the first time, we investigated the selective anti-cancer effects of CD in SH-SY5Y human neuroblastoma cells compared to healthy (normal) fibroblasts (NHDF). Our study revealed selective and dose-dependent cytotoxicity of CD in SH-SY5Y. The natural chalcone CD specifically altered the mitochondrial membrane potential (ΔΨm), as an early marker of apoptosis, in human neuroblastoma cells. Caspase activity was also selectively induced and the amount of cleaved caspase substrates such as PARP was thus increased in human neuroblastoma cells. CD-mediated apoptotic cell death was rescued by pan caspase inhibitor Z-VAD-FMK. The natural chalcone CD selectively induced apoptosis, the programmed cell death, in SH-SY5Y human neuroblastoma cells whereas NHDF being a model for normal (healthy) cells were unaffected. Our data indicates a clinical potential of CD in the more selective and less harmful treatment of neuroblastoma.

EPH115 The Effect of Non-Steroidal Anti-Inflammatory Drugs on C-Reactive Protein Levels and Mortality in Patients with Sepsis

A recent in vitro study found that ibuprofen, naproxen, and ketorolac are competitive caspase inhibitors, a promising anti-inflammatory target for septic shock treatment. However, the impact of such NSAIDs on health outcomes among patients with sepsis is unknown.

Alkaloid from Geissospermum sericeum Benth. & Hook.f. ex Miers (Apocynaceae) Induce Apoptosis by Caspase Pathway in Human Gastric Cancer Cells.

Gastric cancer is among the major causes of death from neoplasia leading causes of death worldwide, with high incidence rates and problems related to its treatment. Here, we outline how Geissospermum sericeum exerts antitumor activity on the ACP02 cell line (human gastric adenocarcinoma) and the mechanism of cell death. The ethanol extract and fractions, neutral fraction and alkaloid fraction, were characterized by thin-layer chromatography and HPLC-DAD, yielding an alkaloid (geissoschizoline N4-methylchlorine) identified by NMR. The cytotoxicity activity of the samples (ethanol extract, neutral fraction, alkaloid fraction, and geissoschizoline N4-methylchlorine) in HepG2 and VERO cells was determined by MTT. The ACP02 cell line was used to assess the anticancer potential. Cell death was quantified with the fluorescent dyes Hoechst 33342, propidium iodide, and fluorescein diacetate. The geissoschizoline N4-methylchlorine was evaluated in silico against caspase 3 and 8. In the antitumor evaluation, there was observed a more significant inhibitory effect of the alkaloid fraction (IC50 18.29 µg/mL) and the geissoschizoline N4-methylchlorine (IC50 12.06 µg/mL). However, geissoschizoline N4-methylchlorine showed lower cytotoxicity in the VERO (CC50 476.0 µg/mL) and HepG2 (CC50 503.5 µg/mL) cell lines, with high selectivity against ACP02 cells (SI 39.47 and 41.75, respectively). The alkaloid fraction showed more significant apoptosis and necrosis in 24 h and 48 h, with increased necrosis in higher concentrations and increased exposure time. For the alkaloid, apoptosis and necrosis were concentration- and time-dependent, with a lower necrosis rate. Molecular modeling studies demonstrated that geissoschizoline N4-methylchlorine could occupy the active site of caspases 3 and 8 energetically favorably. The results showed that fractionation contributed to the activity with pronounced selectivity for ACP02 cells, and geissoschizoline N4-methylchlor is a promising candidate for caspase inhibitors of apoptosis in gastric cancer. Thus, this study provides a scientific basis for the biological functions of Geissospermum sericeum, as well as demonstrates the potential of the geissoschizoline N4-methylchlorine in the treatment of gastric cancer.

EM-2, a natural sesquiterpene lactone from Elephantopus mollis H.B.K., enhanced the sensitivity of breast cancer cells to epirubicin by blocking protective autophagy

BackgroundEM-2, a natural sesquiterpene lactone isolated from Elephantopus mollis H.B.K., showed a good anti-breast cancer effect when combined with epirubicin (EPI). However, its synergistic sensitization mechanism remains unclear. PurposeThis study aimed to determine the therapeutic effect and possible synergistic mechanism of EM-2 with EPI in vivo and in vitro and to provide an experimental basis for the treatment of human breast cancer. MethodsCell proliferation was measured with MTT and colony formation assays. Apoptosis and reactive oxygen species (ROS) levels were examined through flow cytometry, and the expression levels of proteins related to apoptosis, autophagy, endoplasmic reticulum stress, and DNA damage were detected through Western blot analysis. Moreover, the caspase inhibitor Z-VAD-FMK, autophagy inhibitors bafilomycin A1 and chloroquine, ER stress inhibitor 4-phenylbutyric acid, and ROS scavenger N-acetyl cysteine were applied to verify signaling pathways. Breast cancer cell lines were used to evaluate the antitumor functions of EM-2 and EPI in vitro and in vivo. ResultsWe demonstrated that in MDA-MB-231 and SKBR3 cells, the IC50 of EPI combined with EM-2 (IC20) was 37.909 and 33.889 times lower than that of EPI alone, respectively. Further study verified that in EPI-resistant lines (MDA-MB-231/EPI), the IC50 of EPI combined with EM-2 (IC20) was 26.305 times lower than that of EPI alone. Mechanistically, EM-2 could reverse the protective effect of EPI against autophagy in SKBR3 and MDA-MB-231 cells. EM-2 and EPI could trigger ER stress. When EM-2 and EPI were used in combination, ER stress was continuously activated, and ER stress-mediated apoptosis was induced. Meanwhile, EM-2 combined with EPI promoted DNA damage then induced apoptosis. In vivo, the volume of breast cancer xenografts in the combination group was smaller than that in the control, EM-2, and EPI groups. Immunohistochemical experiments demonstrated that the combination of EM-2 and EPI could block autophagy and promote ER stress in vivo. ConclusionEM-2 enhances the sensitivity of MDA-MB-231, SKBR3, and EPI-resistant cells to EPI.

Oxidation of caspase-8 by hypothiocyanous acid enables TNF-mediated necroptosis

Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated invivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation.

Aminoprocalcitonin protects against hippocampal neuronal death via preserving oxidative phosphorylation in refractory status epilepticus

Refractory status epilepticus (RSE) is a neurological emergency where sustaining seizure causes severe neuronal death. Currently, there is no available neuroprotectant effective in RSE. Aminoprocalcitonin (NPCT) is a conserved peptide cleaved from procalcitonin, but its distribution and function in the brain remain enigmatic. Survival of neurons relies on sufficient energy supply. Recently, we found that NPCT was extensively distributed in the brain and had potent modulations on neuronal oxidative phosphorylation (OXPHOS), suggesting that NPCT might be involved in neuronal death by regulating energy status. In the present study, combining biochemical and histological methods, high-throughput RNA-sequence, Seahorse XFe analyser, an array of mitochondria function assays, and behavior-electroencephalogram (EEG) monitoring, we investigated the roles and translational values of NPCT in neuronal death after RSE. We found that NPCT was extensively distributed throughout gray matters in rat brain while RSE triggered NPCT overexpression in hippocampal CA3 pyramidal neurons. High-throughput RNA-sequence demonstrated that the influences of NPCT on primary hippocampal neurons were enriched in OXPHOS. Further function assays verified that NPCT facilitated ATP production, enhanced the activities of mitochondrial respiratory chain complexes I, IV, V, and increased neuronal maximal respiration capacity. NPCT exerted multiple neurotrophic effects including facilitating synaptogenesis, neuritogenesis, spinogenesis, and suppression of caspase-3. A polyclonal NPCT immunoneutralization antibody was developed to antagonize NPCT. In the in vitro 0-Mg2+ seizure model, immunoneutralization of NPCT caused more neuronal death, while exogenous NPCT supplementation, though did not reverse death outcomes, preserved mitochondrial membrane potential. In rat RSE model, both peripheral and intracerebroventricular immunoneutralization of NPCT exacerbated hippocampal neuronal death and peripheral immunoneutralization increased mortality. Intracerebroventricular immunoneutralization of NPCT further led to more serious hippocampal ATP depletion, and significant EEG power exhaustion. We conclude that NPCT is a neuropeptide regulating neuronal OXPHOS. During RSE, NPCT was overexpressed to protect hippocampal neuronal survival via facilitating energy supply.

Cyclophosphamide enfeebles myocardial isometric contraction force via RIP1/RIP3/MLKL/TRPM7-mediated necroptosis

This study explores the negative impact of cyclophosphamide (CP) on cardiac contractility by specifically examining its effect on the active and passive tension of the cardiac muscle in-vitro and revealing the mechanism through which CP induces myocardial insult in-vivo. In young male Sprague-Dawley rats, cardiac toxicity was induced by a single intraperitoneal injection of CP (150 mg/kg body weight). Axial heart tissue slices were electrically stimulated, and the total isometric contraction force was measured at varying pretension levels. Blood and tissue biochemical assays, and histological/ immuno-histological assessments were conducted to evaluate the underlying molecular mechanisms. Statistical analysis shows that there is a significant difference between the drugged and the control groups in terms of the active tension values. Moreover, the pre-tension stress significantly affects both the active and passive tension values. CP altered heart, body, and heart-to-body weight, desolated cardiac muscle architecture, surged cardiac enzymes (CK-MB, LDH, and cTn l), augmented myocardial oxidative stressors (MDA), and weakened myocardial antioxidant status (SOD and GSH). Mechanistically, cyclophosphamide prompted the necroptotic trajectory evidenced by the activation of RIPK1, RIPK3, MLKL and TRPM7, the inhibition of caspase 8 and BCL2 and the upregulation of the protein/mRNA expression of TNF-α and TNFR1. This study identifies necroptosis as a key factor in cyclophosphamide-evoked myocardial contractility impairment, highlighting its potential as a target for alleviating antitumor-related myocardial damage. This innovative approach to investigating the underlying mechanisms of CP-induced cardiac toxicity offers valuable insights into the potential of developing new therapies to mitigate cyclophosphamide’s negative impact.

Hexavalent chromium disrupts the skin barrier by targeting ROS-mediated mitochondrial pathway apoptosis in keratinocytes

Hexavalent chromium (Cr(VI)), a toxic heavy metal, is ubiquitous in daily life. Exposure to this toxic substance in occupational settings can cause dermatitis and cancer. As the body's largest organ, the skin plays a crucial role in protecting the organism against external aggressions. While previous studies have focused on the effects of Cr(VI) on skin inflammation, this study investigates the potential toxicity of Cr(VI) from the skin barrier and integrity perspective. The in vivo results of this study showed that mice exposed to Cr(VI) experienced skin deterioration and hemorrhaging, as well as a reduction in the thickness of the collagen fiber layer. TUNEL and Occludin staining results revealed that Cr(VI)'s toxicity primarily targeted keratinocytes. Experiments in vitro demonstrated that Cr(VI) treatment decreased the activity of HaCaT cells, altered cell morphology, and increased LDH secretion. Further research revealed that Cr(VI) could modify membrane permeability, impair membrane integrity, and reduce the protein expression of ZO-1 and Occludin. In addition, it was discovered that Cr(VI) promoted cell apoptosis and inhibited AKT activation. However, the addition of a caspase inhibitor and an AKT activator prevented Cr(VI)-induced injury to the cell membrane barrier, indicating that apoptosis plays a crucial role in this process. The addition of three apoptotic pathway inhibitors, confirmed that Cr(VI) damaged the cell barrier through ROS-mediated mitochondrial pathway apoptosis. Moreover, the use of a ROS inhibitor significantly reduced Cr(VI)-induced apoptosis and cell barrier injury. In conclusion, this study provides an experimental foundation for the treatment of skin injury caused by Cr(VI).

Inhibition of caspase 1 reduces blood pressure, cytotoxic NK cells, and inflammatory TH17 cells in placental ischemic rats

Preeclampsia (PE) is characterized by maternal hypertension, fetal growth restriction (FGR), and increased inflammation and immune activation, including cytotoxic NK cells (cNKs) and inflammatory T-Helper17 cells (T H 17s). Caspase 1 activity directly leads to release of the inflammatory cytokines IL1β and IL18. Using the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia, we tested the hypothesis that inhibition of caspase 1 with Belnacasan in RUPP rats will attenuate PE pathophysiology. On gestation day (GD) 14, timed pregnant 10–12-week-old Sprague Dawley rats underwent the RUPP or Sham procedure and received either vehicle or Belnacasan (50mg/kg) daily i.p. from GD14 through GD19: RUPP + vehicle, RUPP + Belnacasan, Sham + vehicle, Sham + Belnacasan. Carotid catheters were placed on GD18. On GD19, MAP was measured, animals were euthanized, and blood and tissues were collected for analysis. Bioplex and flow cytometry analysis were performed on placental tissues. Placental IL1β was markedly increased in RUPP rats vs. Sham rats (44.2 vs. 21.67 pg/mg, p=0.0009). Belnacasan treatment significantly reduced IL1β in RUPP rats (21.90 pg/mg; p=0.0006 vs RUPP). Likewise, placental IL18 was increased in RUPP rats vs Sham (27.1 vs 6.7 pg/mg, p=0.007), and inhibition of caspase 1 reduced RUPP IL18 (6.2, p=0.01 vs RUPP) to Sham levels. No significant differences in IL1β or IL18 were observed in treated Sham rats. These data show the administered Belnacasan dose is effective against Caspase 1 activity. Caspase 1 inhibition reduced placental cNKs in treated-RUPP vs control RUPP (0.155% vs. 0.362 % gated; p=0.043). Belnacasan treatment also reduced placental T H 17s in RUPP rats vs. control RUPPs (1.053% vs 7.25% gated; p=0.003). Increased MAP was observed in RUPP rats vs Sham (131.5 mmHg vs 104.8 mmHg; p<0.0001) and was reduced in RUPP + Belnacasan (117.5 mmHg; p=0.0016 vs RUPP). No changes were observed in MAP of Sham rats treated with Belnacasan. Placental reactive oxygen species (ROS) were elevated in RUPP vs Sham (456.4 vs 198.8 RLU/min/mg, p=0.029). Caspase 1 inhibition significantly reduced ROS in treated RUPP rats (210.3, p=0.031) vs RUPP), and had no effect on ROS in treated Sham rats (156.3). Caspase 1 inhibition had no effect on fetal weight or placental efficiency in any groups. Additional analyses will include assessment of other cytokines in blood and tissue, as well as vascular function in all groups. In conclusion, caspase 1 inhibition reduces placental T H 17 and cNK populations and reduces MAP in RUPP rats. These data suggest that caspase 1 is a key contributor to PE pathophysiology and may represent a therapeutic target to improve maternal outcomes in PE. This work was supported by National Institutes of Health Grants T32HL105324 to C. A. Shields, F31HL165852 to Xi Wang, and R01HL151407 to D. C. Cornelius. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Baicalin inhibits influenza A (H1N1)-induced pyroptosis of lung alveolar epithelial cells via caspase-3/GSDME pathway.

Baicalin (7-d-glucuronic acid-5, 6-dihydroxyflavone) derived from the root of Scutellaria baicalensis used as Traditional Chinese Medicine (TCM) has been revealed to exert potential antiviral activity via various pathways, while the molecular mechanisms have not been fully understood. Pyroptosis, an inflammatory form of programmed cell death (PCD), is reported to play a crucial role in host cell fate during viral infection. In this study, transcriptome analysis of mice lung tissue reveals that baicalin reverses the alterations of the mRNA levels of PCD-associated genes upon H1N1 challenge, with a concomitant decrease in the population of H1N1-induced propidium iodide (PI)+ and Annexin Ⅴ+ cells. Intriguingly, we find that baicalin contributes to the survival of infected lung alveolar epithelial cells partly through its inhibition of H1N1-induced cell pyroptosis, which is manifested by reduced bubble-like protrusion cells and lactate dehydrogenase (LDH) release. Moreover, the antipyroptosis effect of baicalin in response to H1N1 infection is found to be mediated by its repression on caspase-3/Gasdermin E (GSDME) pathway. Cleaved caspase-3 and N-terminal fragment of GSDME (GSDME-N) are detected in H1N1-infected cell lines and mice lung tissues, which are markedly reversed by baicalin treatment. Furthermore, inhibition of caspase-3/GSDME pathway by caspase-3 inhibitor or siRNA exerts an antipyroptosis effect equal to that of baicalin treatment in infected A549 and BEAS-2B cells, indicating a pivotal role of caspase-3 in the antiviral activities of baicalin. Conclusively, for the first time, we demonstrate that baicalin could effectively suppress H1N1-induced pyroptosis of lung alveolar epithelial cells via caspase-3/GSDME pathway both in vitro and in vivo.

In Silico Evaluation: Bioactive Compounds in Soursop Plant (Annona muricata L.) as Caspase-3 Inhibitor for Prostate Cancer

Background: Prostate cancer has become one of the leading causes of death in men. Cancer patients often seek alternative treatments apart from chemotherapy, radiation therapy, and surgery. The use of medicinal plants in both preventive and curative actions in healthcare has been widely recognized. One of the plants known to have anticancer activity is the soursop leaf (Annona muricata L.). Objective: This study was conducted to explore the potential of active compounds contained in A. muricata as drug candidates for the inhibition of caspase-3 in silico. Methods: The research began with the prediction of Lipinski's Rule of Five and ADMET properties for the compounds found in A. muricata. The prediction process was followed by pharmacophore modelling and molecular docking simulations on caspase-3 (PDB: 1NME) as the target protein and 2-hydroxy-5-(2-mercapto-ethylsulfamoyl)-benzoic acid as the natural ligand using AutoDockTools 1.5.6. Results: Based on the molecular docking results, 22 test ligands were able to form bonds with the caspase-3 enzyme. The two best interactions were observed with the test ligands, Isolaureline and S-norcorydine, with binding energy values of -6.20 kcal /mol and -6.12 kcal /mol and inhibition constant values of 28.65 µM and 32.53 µM. In terms of receptor-target interactions, these two compounds also exhibited hydrogen bonding and van der Waals interactions similar to the natural ligand. Conclusion: The best bioactive compounds in A. muricata (Isolaureline and S-norcorydine) were predicted to have the ability to interact with caspase-3 and the potential to be used as prostate cancer drug candidates.

Engaging a Non-catalytic Cysteine Residue Drives Potent and Selective Inhibition of Caspase-6.

Caspases are a family of cysteine-dependent proteases with important cellular functions in inflammation and apoptosis, while also implicated in human diseases. Classical chemical tools to study caspase functions lack selectivity for specific caspase family members due to highly conserved active sites and catalytic machinery. To overcome this limitation, we targeted a non-catalytic cysteine residue (C264) unique to caspase-6 (C6), an enigmatic and understudied caspase isoform. Starting from disulfide ligands identified in a cysteine trapping screen, we used a structure-informed covalent ligand design to produce potent, irreversible inhibitors (3a) and chemoproteomic probes (13-t) of C6 that exhibit unprecedented selectivity over other caspase family members and high proteome selectivity. This approach and the new tools described will enable rigorous interrogation of the role of caspase-6 in developmental biology and in inflammatory and neurodegenerative diseases.

DNA methyltransferase inhibitor 5-azacytidine enhances neuroblastoma cell lysis by an oncolytic parainfluenza virus.

Studies with neuroblastoma have shown that the presence of aberrant DNA epigenetic modifications mediated by DNA methyltransferases correlates with poor prognosis, making these enzymes a target for therapeutics based on synthetic epigenetic modulators such as DNA methyltransferase inhibitors (DNMTi). Here, we have used a neuroblastoma cell line model to test the hypothesis that treatment with a DNMTi would enhance cell killing when used in combination with oncolytic Parainfluenza virus 5 (P/V virus), a cytoplasmic-replicating RNA virus. Pretreatment of SK-N-AS cells with the DNMTi 5-azacytidine substantially enhanced P/V virus-mediated cell death in a dose- and multiplicity of infection-dependent manner. Infection with the virus alone and the combination treatment with 5-azacytidine and P/V virus infection led to the activation of caspases-8, -9, and -3/7. Inhibition of caspases using a pan-caspase inhibitor minimally affected cell killing by P/V virus alone, but by contrast, largely reduced cell death mediated by 5-azacytidine treatment alone or in combination with P/V virus infection. 5-Azacytidine pretreatment dampened P/V virus gene expression and growth within the SK-N-AS cell population, which correlated with enhanced expression of important antiviral genes such as interferon-β and OAS2 . Taken together, our data support the role of combination treatment using 5-azacytidine and an oncolytic P/V virus for neuroblastoma therapy.