Caspase Activation Research Articles

Non-canonical autophosphorylation of RIPK1 drives timely pyroptosis to control Yersinia infection

Caspase-8-dependent pyroptosis has been shown to mediate host protection from Yersinia infection. For this mode of cell death, the kinase activity of receptor-interacting protein kinase 1 (RIPK1) is required, but the autophosphorylation sites required to drive caspase-8 activation have not been determined. Here, we show that non-canonical autophosphorylation of RIPK1 at threonine 169 (T169) is necessary for caspase-8-mediated pyroptosis. Mice with alanine in the T169 position are highly susceptible to Yersinia dissemination. Mechanistically, the delayed formation of a complex containing RIPK1, ZBP1, Fas-associated protein with death domain (FADD), and caspase-8 abrogates caspase-8 maturation in T169A mice and leads to the eventual activation of RIPK3-dependent necroptosis invivo; however, this is insufficient to protect the host, suggesting that timely pyroptosis during early response is specifically required to control infection. These results position RIPK1 T169 phosphorylation as a driver of pyroptotic cell death critical for host defense.

CHCHD2 P14L, found in amyotrophic lateral sclerosis, exhibits cytoplasmic mislocalization and alters Ca2+ homeostasis.

CHCHD2 and CHCHD10, linked to Parkinson's disease and amyotrophic lateral sclerosis-frontotemporal dementia (ALS), respectively, are mitochondrial intermembrane proteins that form a heterodimer. This study aimed to investigate the impact of the CHCHD2 P14L variant, implicated in ALS, on mitochondrial function and its subsequent effects on cellular homeostasis. The missense variant of CHCHD2, P14L, found in a cohort of patients with ALS, mislocalized CHCHD2 to the cytoplasm, leaving CHCHD10 in the mitochondria. Drosophila lacking the CHCHD2 ortholog exhibited mitochondrial degeneration. In contrast, human CHCHD2 P14L, but not wild-type human CHCHD2, failed to suppress this degeneration, suggesting that P14L is a pathogenic variant. The mitochondrial Ca2+ buffering capacity was reduced in Drosophila neurons expressing human CHCHD2 P14L. The altered Ca2+-buffering phenotype was also observed in cultured human neuroblastoma SH-SY5Y cells expressing CHCHD2 P14L. In these cells, transient elevation of cytoplasmic Ca2+ facilitated the activation of calpain and caspase-3, accompanied by the processing and insolubilization of TDP-43. These observations suggest that CHCHD2 P14L causes abnormal Ca2+ dynamics and TDP-43 aggregation, reflecting the pathophysiology of ALS.

Extramacrochaetae regulates Notch signaling in the Drosophila eye through non-apoptotic caspase activity.

Many cell fate decisions are determined transcriptionally. Accordingly, some fate specification is prevented by Inhibitor of DNA binding (Id) proteins that interfere with DNA binding by master regulatory transcription factors. We show that the Drosophila Id protein Extra macrochaetae (Emc) also affect developmental decisions by regulating caspase activity. Emc, which prevents proneural bHLH transcription factors from specifying neural cell fate, also prevents homodimerization of another bHLH protein, Daughterless (Da), and thereby maintains expression of the Death-Associated Inhibitor of Apoptosis (diap1) gene. We found that multiple effects of emc mutations on cell growth and on eye development were all caused by reduced Diap1 levels and corresponding activation of caspases. These effects included acceleration of the morphogenetic furrow, failure of R7 photoreceptor cell specification, and delayed differentiation of non-neuronal cone cells. Within emc mutant clones, Notch signaling was elevated in the morphogenetic furrow, increasing morphogenetic furrow speed. This was associated with caspase-dependent increase in levels of Delta protein, the transmembrane ligand for Notch. Posterior to the morphogenetic furrow, elevated Delta cis-inhibited Notch signaling that was required for R7 specification and cone cell differentiation. Thus, emc mutations reveal the importance of restraining caspase activity even in non-apoptotic cells to prevent abnormal development, in the Drosophila eye through effects on Notch signaling.

Ellagic acid supplementation ameliorates cisplatin-induced liver injury in mice by inhibiting the NF-κB pathway and activating the Nrf2/HO-1 pathway

Cisplatin (cis-diaminodichloroplatinum II, CDDP), an essential chemotherapeutic agent, can cause potential hepatotoxicity, but the underlying molecular mechanisms remain unclear. In this study, the protective effects of ellagic acid (EA) on CDDP exposure-induced hepatotoxicity and the underlying molecular mechanisms were investigated in a mouse model. Mice were randomly divided into control, CDDP model, EA100 (i.e., 100 mg/kg/day), and CDDP plus 25, 50, or 100 mg/kg/day EA groups. Mice in all the CDDP-treated groups were intraperitoneally injected with 20 mg/kg/day CDDP for two days. For all EA cotreatments, the mice were orally administered EA for seven days. Our results revealed that CDDP treatment resulted in liver dysfunction, oxidative stress, and caspase activation, which were effectively attenuated by EA cotreatment in a dose-dependent manner. Furthermore, EA supplementation significantly downregulated the CDDP exposure-induced protein and mRNA expression of NF-κB, IL-1β, TNF-α, and IL-6 but further upregulated the protein and mRNA expression of Nrf2 and HO-1. Molecular docking analysis revealed strong interactions between EA and the NF-κB or Keap1 proteins. In conclusion, our results revealed that EA supplementation could ameliorate CDDP-induced liver toxicity in mice by activating the Nrf2/HO-1 signaling pathway and inhibiting the NF-kB signaling pathway.

Strong activation of p53 by actinomycin D and nutlin-3a overcomes the resistance of cancer cells to the pro-apoptotic activity of the FAS ligand

The FAS ligand (FASLG) is expressed on lymphocytes, which employ it to activate death receptors on target cells. Cancer cells are generally resistant to apoptosis triggered by FASLG. In this work, we found a way to circumvent this resistance by treatment with actinomycin D (ActD) and nutlin-3a (Nut3a). We selected this drug combination based on our transcriptomic data showing strong activation of proapoptotic genes, including those for receptor-mediated apoptosis, in cells exposed to actinomycin D and nutlin-3a. To test our hypothesis, we pre-exposed cancer cell lines to this drug combination for 45 h and then treated them with recombinant FASLG. This almost instantaneously killed most cells. Actinomycin D and nutlin-3a strongly cooperated in the sensitization because the effect of the drugs acting solo was not as spectacular as the drug combination, which together with FASLG killed more than 99% of cells. Based on the caspase activation pattern (caspase-8, caspase-9, caspase-10), we conclude that both extrinsic and intrinsic pro-apoptotic pathways were engaged. In engineered p53-deficient cells, this pro-apoptotic effect was completely abrogated. Therefore, the combination of ActD + Nut3a activates p53 in an extraordinary way, which overcomes the resistance of cancer cells to apoptosis triggered by FASLG. Interestingly, other combinations of drugs, e.g., etoposide + nutlin-3a, actinomycin D + RG7112, and actinomycin D + idasanutlin had a similar effect. Moreover, normal human fibroblasts are less sensitive to death induced by ActD + Nut3a + FASLG. Our findings create the opportunity to revive the abandoned attempts of cancer immunotherapy employing the recombinant FAS ligand.Graphical

Biological Properties of Boletus edulis Extract on Caco-2 Cells: Antioxidant, Anticancer, and Anti-Inflammatory Effects.

Boletus edulis (BE) is a mushroom well known for its taste, nutritional value, and medicinal properties. The objective of this work was to study the biological effects of BE extracts on human colon carcinoma cells (Caco-2), evaluating parameters related to oxidative stress and inflammation. In this study, a hydroethanolic extract of BE was obtained by ohmic heating green technology. The obtained BE extracts are mainly composed of sugars (mainly trehalose), phenolic compounds (taxifolin, rutin, and ellagic acid), and minerals (K, P, Mg, Na, Ca, Zn, Se, etc.). The results showed that BE extracts were able to reduce cancer cell proliferation by the induction of cell cycle arrest at the G0/G1 stage, as well as cell death by autophagy and apoptosis, the alteration of mitochondrial membrane potential, and caspase-3 activation. The extracts modified the redox balance of the cell by increasing the ROS levels associated with a decrease in the thioredoxin reductase activity. Similarly, BE extracts attenuated Caco-2 inflammation by reducing both iNOS and COX-2 mRNA expression and COX-2 protein expression. In addition, BE extracts protected the intestine from the oxidative stress induced by H2O2. Therefore, this study provides information on the potential use of BE bioactive compounds as anticancer therapeutic agents and as functional ingredients to prevent oxidative stress in the intestinal barrier.

Release of IL-1β and IL-18 in human primary bronchial epithelial cells exposed to cigarette smoke is independent of NLRP3.

Cigarette smoke (CS) is a major risk factor for chronic lung diseases and promotes activation of pattern recognition receptors in the bronchial epithelium. NOD-like receptor family, pyrin domain-containing 3 (NLRP3) is a pattern recognition receptor whose activation leads to caspase-1 cleavage, maturation/release of IL-1β and IL-18, and eventually pyroptosis. Whether the NLRP3 inflammasome participates in CS-induced inflammation in bronchial epithelial cells is still unclear. Herein, we evaluated the involvement of NLRP3 in CS-induced inflammatory responses in human primary bronchial epithelial cells. To this purpose, human primary bronchial epithelial cells were stimulated with CS extracts (CSE) and lytic cell death, caspase activation (-1, -8, -3/7), cytokine release (IL-1β, IL-18, and IL-8), NLRP3, pro-IL-1β/pro-IL-18 mRNA, and protein expression were measured. The impact of inhibitors of NLRP3 (MCC950), caspases, and the effect of the antioxidant N-acetyl cysteine were evaluated. We found that CSE increased pro-IL-1β expression and induced activation of caspase-1 and release of IL-1β and IL-18. These events were independent of NLRP3 and we found that NLRP3 was not expressed. N-acetyl cysteine reverted CSE-induced caspase-1 activation. Overall, our findings support that the bronchial epithelium may play a central role in the release of IL-1 family cytokines independently of NLRP3 in the lungs of smokers.

Regeneration following tissue necrosis is mediated by non-apoptotic caspase activity.

Tissue necrosis is a devastating complication for many human diseases and injuries. Unfortunately, our understanding of necrosis and how it impacts surrounding healthy tissue - an essential consideration when developing methods to treat such injuries - has been limited by a lack of robust genetically tractable models. Our lab previously established a method to study necrosis-induced regeneration in the Drosophila wing imaginal disc, which revealed a unique phenomenon whereby cells at a distance from the injury upregulate caspase activity in a process called Necrosis-induced Apoptosis (NiA) that is vital for regeneration. Here we have further investigated this phenomenon, showing that NiA is predominantly associated with the highly regenerative pouch region of the disc, shaped by genetic factors present in the presumptive hinge. Furthermore, we find that a proportion of NiA fail to undergo apoptosis, instead surviving effector caspase activation to persist within the tissue and stimulate reparative proliferation late in regeneration. This proliferation relies on the initiator caspase Dronc, and occurs independent of JNK, ROS or mitogens associated with the previously characterized Apoptosis-induced Proliferation (AiP) mechanism. These data reveal a new means by which non-apoptotic Dronc signaling promotes regenerative proliferation in response to necrotic damage.

Pioglitazone ameliorates sepsis-associated encephalopathy through SIRT1 signaling pathway

Sepsis is a severe immune response to an infection. It is associated with multiple organ dysfunction syndrome (MODs) along with systemic and neuronal inflammatory response. This study focused on the acute neurologic dysfunction associated with sepsis by exploring the role of PPARγ/SIRT1 pathway against sepsis. We studied the role of this axis in ameliorating sepsis-associated encephalopathy (SAE) and its linked neurobehavioral disorders by using pioglitazone (PIO). This PPARγ agonist showed neuroprotective actions in neuroinflammatory disorders. Sepsis was induced in mice by LPS (10 mg/kg). Survival rate and MODs were assessed. Furthermore, behavioral deficits, cerebral oxidative, inflammatory, and apoptotic markers, and the cerebral expression level of SIRT1 were determined. In this study, we observed that PIO attenuated sepsis-induced cerebral injury. PIO significantly enhanced survival rate, attenuated MODs, and systemic inflammatory response in septic mice. PIO also promoted cerebral SIRT1 expression and reduced cerebral activation of microglia, oxidative stress, HMGB, iNOS, NLRP3 and caspase-3 along with an obvious improvement in behavioral deficits and cerebral pathological damage induced by LPS. Most of the neuroprotective effects of PIO were abolished by EX-527, a SIRT1 inhibitor. These results highlight that the neuroprotective effect of PIO in SAE is mainly SIRT1-dependent.

Distinct sequential death complexes regulate pyroptosis and IL-1β release in response to Yersinia blockade of immune signaling.

Pathogen infection of host cells triggers an inflammatory cell death termed pyroptosis via activation of inflammatory caspases. However, blockade of immune signaling kinases by the Yersinia virulence factor YopJ triggers cell death involving both apoptotic caspase-8 and pyroptotic caspase-1. While caspase-1 is normally activated within inflammasomes, Yersinia-induced caspase-1 activation is independent of known inflammasome components. We report that caspase-8 is an essential initiator, while caspase-1 is an essential amplifier of its own activation through two feed-forward loops involving caspase-1 auto-processing and caspase-1-dependent activation of gasdermin D and NLPR3. Notably, while Yersinia-induced caspase-1 activation and cell death are inflammasome-independent, IL-1β release requires NLPR3 inflammasome activation. Mechanistically, caspase-8 is rapidly activated within multiple foci throughout the cell, followed by assembly of a canonical inflammasome speck, indicating that caspase-8 and canonical inflammasome complex assemblies are kinetically and spatially distinct. Our findings reveal that functionally interconnected but distinct death complexes mediate pyroptosis and IL-1β release in response to pathogen blockade of immune signaling.

Allicin: a promising modulator of apoptosis and survival signaling in cancer.

According to the World Health Organization, cancer is the foremost cause of mortality globally. Various phytochemicals from natural sources have been extensively studied for their anticancer properties. Allicin, a powerful organosulfur compound derived from garlic, exhibits anticancer, antioxidant, anti-inflammatory, antifungal, and antibacterial properties. This review aims to update and evaluate the chemistry, composition, mechanisms of action, and pharmacokinetics Allicin. Allicin has garnered significant attention for its potential role in modulating Fas-FasL, Bcl2-Bax, PI3K-Akt-mTOR, autophagy, and miRNA pathways. At the molecular level, allicin induces the release of cytochrome c from the mitochondria and enhances the activation of caspases-3, -8, and -9. This is accompanied by the simultaneous upregulation of Bax and Fas expression in tumor cells. Allicin can inhibit excessive autophagy by activating the PI3K/Akt/mTOR and MAPK/ERK/mTOR signaling pathways. Allicin-loaded nano-formulations efficiently induce apoptosis in cancer cells while minimizing toxicity to normal cells. Safety and clinical aspects are meticulously scrutinized, providing insights into the tolerability and adverse effects associated with allicin administration, along with an overview of current clinical trials evaluating its therapeutic potential. In conclusion, this review underscores the promising prospects of allicin as a dietary-derived medicinal compound for cancer therapy. It emphasizes the need for further research to elucidate its precise mechanisms of action, optimize delivery strategies, and validate its efficacy in clinical settings.

Crotoxin induces cytotoxic effects in human malignant melanoma cells in both native and detoxified forms.

Introduction: Melanoma, a highly aggressive skin cancer originating in melanocytes, poses a significant threat due to its metastatic potential. While progress has been made in treating melanoma with targeted therapies and immunotherapies, challenges persist. Crotoxin (CTX), the principal toxin in Crotalus durissus terrificus snake venom, exhibits various biological activities, including anti-tumoral effects across multiple cancers. However, its clinical use is limited by toxicity. Thus, exploring alternatives to mitigate adverse effects is crucial. Methods and Results: This study investigates the antitumoral potential of CTX in its native and in a detoxified form, in melanoma cells. Firstly, we demonstrated that detoxified CTX presented reduced phospholipase activity. Both forms proved to be more cytotoxic to SK-MEL-28 and MeWo melanoma cells than non-tumoral cells. In SK-MEL-28 cells, where cytotoxic effects were more pronounced, native and detoxified CTX induced increased necrosis and apoptosis rates. We also confirmed the apoptosis death demonstrated by the activation of caspase-3 and 7, and the formation of apoptotic bodies. Furthermore, both CTX caused cell cycle arrest at the G2/M phase, interfering with melanoma cell proliferation. Cell migration and invasion were also suppressed by both CTX. These results confirm the antitumoral potential of CTX. Discussion: The maintenance of the antiproliferative effects in the detoxified version, with reduced enzymatic activity often liked to harm effects, supports further studies to identify active parts of the molecule responsible for the interesting effects without causing substantial toxic events, contributing to the future use of CTX-derived drugs with safety and efficacy.

Evaluation of effects of bisphenol analogs AF, S, and F on viability, proliferation, production of selected cancer-related factors, and expression of selected transcripts in Caov-3 human ovarian epithelial cell line

Bisphenol A (BPA) has been a substantial additive in plastics until the reports on its adverse effects have led to its restrictions and replacement. Monitoring studies document the increasing occurrence of bisphenol analogs, however, data on their effects and risks is still insufficient. Based on the indications that BPA might contribute to ovarian cancer pathogenesis, we examined effects of the analogs AF (BPAF), S (BPS) and F (BPF) (10−9–10−4 M) on the Caov-3 epithelial cancer cells, including the impact on cell viability, proliferation, oxidative stress, and production and expression of several factors and genes related to ovarian cancer. At environmentally relevant doses, bisphenols did not exert significant effects. At the highest concentration, BPAF caused varied alterations, including decreased cell viability and proliferation, caspase activation, down-regulation of PCNA and BIRC5, elevation of IL8, VEGFA, MYC, PTGS2 and ABCB1 expressions. Only BPA (10−4 M) increased IL-6, IL-8 and VEGFA output by the Caov-3 cells. Each bisphenol induced generation of reactive oxygen species and decreased superoxide dismutase activity at the highest concentration. Although the effects were observed only in the supraphysiological doses, the results indicate that certain bisphenol analogs might affect several ovarian cancer cell characteristics and merit further investigation.

Novel Thienopyrimidine-Hydrazinyl Compounds Induce DRP1-Mediated Non-Apoptotic Cell Death in Triple-Negative Breast Cancer Cells.

Apoptosis induction with taxanes or anthracyclines is the primary therapy for TNBC. Cancer cells can develop resistance to anticancer drugs, causing them to recur and metastasize. Therefore, non-apoptotic cell death inducers could be a potential treatment to circumvent apoptotic drug resistance. In this study, we discovered two novel compounds, TPH104c and TPH104m, which induced non-apoptotic cell death in TNBC cells. These lead compounds were 15- to 30-fold more selective in TNBC cell lines and significantly decreased the proliferation of TNBC cells compared to that of normal mammary epithelial cell lines. TPH104c and TPH104m induced a unique type of non-apoptotic cell death, characterized by the absence of cellular shrinkage and the absence of nuclear fragmentation and apoptotic blebs. Although TPH104c and TPH104m induced the loss of the mitochondrial membrane potential, TPH104c- and TPH104m-induced cell death did not increase the levels of cytochrome c and intracellular reactive oxygen species (ROS) and caspase activation, and cell death was not rescued by incubating cells with the pan-caspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). Furthermore, TPH104c and TPH104m significantly downregulated the expression of the mitochondrial fission protein, DRP1, and their levels determined their cytotoxic efficacy. Overall, TPH104c and TPH104m induced non-apoptotic cell death, and further determination of their cell death mechanisms will aid in the development of new potent and efficacious anticancer drugs to treat TNBC.

Synergistic Enhancement of Chemotherapy-Induced Cell Death and Antitumor Efficacy against Tumoral T-Cell Lymphoblasts by IMMUNEPOTENT CRP.

T-cell malignancies, including T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL), present significant challenges to treatment due to their aggressive nature and chemoresistance. Chemotherapies remain a mainstay for their management, but the aggressiveness of these cancers and their associated toxicities pose limitations. Immunepotent CRP (ICRP), a bovine dialyzable leukocyte extract, has shown promise in inducing cytotoxicity against various cancer types, including hematological cancers. In this study, we investigated the combined effect of ICRP with a panel of chemotherapies on cell line models of T-ALL and T-LBL (CEM and L5178Y-R cells, respectively) and its impact on immune system cells (peripheral blood mononuclear cells, splenic and bone marrow cells). Our findings demonstrate that combining ICRP with chemotherapies enhances cytotoxicity against tumoral T-cell lymphoblasts. ICRP + Cyclophosphamide (CTX) cytotoxicity is induced through a caspase-, reactive oxygen species (ROS)-, and calcium-dependent mechanism involving the loss of mitochondrial membrane potential, an increase in ROS production, and caspase activation. Low doses of ICRP in combination with CTX spare non-tumoral immune cells, overcome the bone marrow-induced resistance to CTX cell death, and improves the CTX antitumor effect in vivo in syngeneic Balb/c mice challenged with L5178Y-R. This led to a reduction in tumor volume and a decrease in Ki-67 proliferation marker expression and the granulocyte/lymphocyte ratio. These results set the basis for further research into the clinical application of ICRP in combination with chemotherapeutic regimens for improving outcomes in T-cell malignancies.

IPSC-induced neurons with the V337M MAPT mutation are selectively vulnerable to caspase-mediated cleavage of tau and apoptotic cell death

BackgroundTau post-translational modifications (PTMs) result in the gradual build-up of abnormal tau and neuronal degeneration in tauopathies, encompassing variants of frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD). Tau proteolytically cleaved by active caspases, including caspase-6, may be neurotoxic and prone to self-aggregation. Also, our recent findings show that caspase-6 truncated tau represents a frequent and understudied aspect of tau pathology in AD in addition to phospho-tau pathology. In AD and Pick's disease, a large percentage of caspase-6 associated cleaved-tau positive neurons lack phospho-tau, suggesting that many vulnerable neurons to tau pathology go undetected when using conventional phospho-tau antibodies and possibly will not respond to phospho-tau based therapies. Therefore, therapeutic strategies against caspase cleaved-tau pathology could be necessary to modulate the extent of tau abnormalities in AD and other tauopathies. MethodsTo understand the timing and progression of caspase activation, tau cleavage, and neuronal death, we created two mAbs targeting caspase-6 tau cleavage sites and probed postmortem brain tissue from an individual with FTLD due to the V337M MAPT mutation. We then assessed tau cleavage and apoptotic stress response in cortical neurons derived from induced pluripotent stem cells (iPSCs) carrying the FTD-related V337M MAPT mutation. Finally, we evaluated the neuroprotective effects of caspase inhibitors in these iPSC-derived neurons. ResultsFTLD V337M MAPT postmortem brain showed positivity for both cleaved tau mAbs and active caspase-6. Relative to isogenic wild-type MAPT controls, V337M MAPT neurons cultured for 3 months post-differentiation showed a time-dependent increase in pathogenic tau in the form of caspase-cleaved tau, phospho-tau, and higher levels of tau oligomers. Accumulation of toxic tau species in V337M MAPT neurons was correlated with increased vulnerability to pro-apoptotic stress. Notably, this mutation-associated cell death was pharmacologically rescued by the inhibition of effector caspases. ConclusionsOur results suggest an upstream, time-dependent accumulation of caspase-6 cleaved tau in V337M MAPT neurons promoting neurotoxicity. These processes can be reversed by caspase inhibition. These results underscore the potential of developing caspase-6 inhibitors as therapeutic agents for FTLD and other tauopathies. Additionally, they highlight the promise of using caspase-cleaved tau as biomarkers for these conditions.

Activation of p38 and JNK by ROS Contributes to Deoxybouvardin-Mediated Intrinsic Apoptosis in Oxaliplatin-Sensitive and -Resistant Colorectal Cancer Cells.

Colorectal cancer (CRC) remains a global health burden, accounting for almost a million deaths annually. Deoxybouvardin (DB), a non-ribosomal peptide originally isolated from Bouvardia ternifolia, has been reported to possess antitumor activity; however, the detailed mechanisms underlying this anticancer activity have not been elucidated. We investigated the anticancer activity of the cyclic hexapeptide, DB, in human CRC HCT116 cells. Cell viability, evaluated by MTT assay, revealed that DB suppressed the growth of both oxaliplatin (Ox)-resistant HCT116 cells (HCT116-OxR) and Ox-sensitive cells in a concentration- and time-dependent manner. Increased reactive oxygen species (ROS) generation was observed in DB-treated CRC cells, and it induced cell cycle arrest at the G2/M phase by regulating p21, p27, cyclin B1, and cdc2 levels. In addition, Western blot analysis revealed that DB activated the phosphorylation of JNK and p38 MAPK in CRC. Furthermore, mitochondrial membrane potential (MMP) was dysregulated by DB, resulting in cytochrome c release and activation of caspases. Taken together, DB exhibited anticancer activity against both Ox-sensitive and Ox-resistant CRC cells by targeting JNK and p38 MAPK, increasing cellular ROS levels, and disrupting MMP. Thus, DB is a potential therapeutic agent for the treatment of Ox-resistant CRC.

Lipopolysaccharide-Induced Lysosomal Cell Death Through Reactive Oxygen Species in Rat Liver Cell Clone 9.

In sepsis, bacterial components, particularly lipopolysaccharide (LPS), trigger organ injuries such as liver dysfunction. Although sepsis induces hepatocyte damage, the mechanisms underlying sepsis-related hepatic failure remain unclear. In this study, we demonstrated that the LPS-treated rat hepatocyte cell line Clone 9 not only induced reactive oxygen species (ROS) generation and apoptosis but also increased the expression of the autophagy marker proteins LC3-II and p62, and decreased the expression of intact Lamp2A, a lysosomal membrane protein. Additionally, LPS increased lysosomal membrane permeability and galectin-3 puncta formation, and promoted lysosomal alkalization in Clone 9 cells. Pharmacological inhibition of caspase-8 and cathepsin D (CTSD) suppressed the activation of caspase-3 and rescued the viability of LPS-treated Clone 9 cells. Furthermore, LPS induced CTSD release associated with lysosomal leakage and contributed to caspase-8 activation. Pretreatment with the antioxidant N-acetylcysteine (NAC) not only diminished ROS generation and increased the cell survival rate, but also decreased the expression of activated caspase-8 and caspase-3 and increased the protein level of Lamp2A in LPS-treated Clone 9 cells. These results demonstrate that LPS-induced ROS causes lysosomal membrane permeabilization and lysosomal cell death, which may play a crucial role in hepatic failure in sepsis. Our results may facilitate the development of new strategies for sepsis management.

JWH133 attenuates behavior deficits and iron accumulation in 6-OHDA-induced Parkinson's disease model rats.

Growing evidence indicates that activation of cannabinoid type 2 (CB2) receptors protects dopamine neurons in the pathogenesis of Parkinson's disease (PD). However, the mechanisms underlying neuroprotection mediated by CB2 receptors are still elusive. In this study, we investigated the effects of CB2 receptor activation on 6-hydroxydopamine (6-OHDA)-induced dopamine neuron degeneration and iron accumulation in the substantia nigra (SN) of rats. We found that treatment with JWH133, a selective CB2 receptor agonist, significantly improved the apomorphine (APO)-induced rotational behavior in 6-OHDA-treated rats. The decreased numbers of tyrosine hydroxylase (TH)-positive neurons and reduced TH protein expression in the lesioned SN of rats were effectively restored by JWH133. Moreover, we found that JWH133 inhibited the increase of iron-staining cells in the lesioned SN of rats. To explore the protective mechanisms of activation of CB2 receptors on dopamine neurons, we further observed the effect of JWH133 on 1-methyl-4-phenylpyridinium (MPP+)-treated primary cultured ventral mesencephalon (VM) neurons from rats. We found that JWH133 significantly inhibited the increase of intracellular reactive oxygen species (ROS), the activation of Caspase-3, the decrease of mitochondrial transmembrane potential (ΔΨm), and the decrease of Bcl-2/Bax protein expression caused by MPP+ treatment. JWH133 also inhibited the MPP+-induced upregulation of divalent metal transporter-1 (DMT1) and downregulation of ferroportin 1 (FPN1). Furthermore, JWH133 also suppressed the MPP+-accelerated iron influx in the VM neurons. These results suggest that activation of CB2 receptor suppresses MPP+-induced cellular iron accumulation and prevents neurodegeneration.NEW & NOTEWORTHY Expression of cannabinoid type 2 receptors (CB2Rs) was discovered on dopamine neurons in recent years. The role of CB2R expressed on dopamine neurons in the pathogenesis of Parkinson's disease (PD) has not been fully elucidated. The content of iron accumulation in the brain is closely related to the progress of PD. We verified the inhibitory effect of CB2R on iron deposition in dopamine neurons through experiments, which provided a new idea for the treatment of PD.

ROS-responsive charge reversal mesoporous silica nanoparticles as promising drug delivery system for neovascular retinal diseases

Intravitreal injection of biodegradable implant drug carriers shows promise in reducing the injection frequency for neovascular retinal diseases. However, current intravitreal ocular devices have limitations in adjusting drug release rates for individual patients, thereby affecting treatment effectiveness. Accordingly, we developed mesoporous silica nanoparticles (MSNs) featuring a surface that reverse its charge in response to reactive oxygen species (ROS) for efficient delivery of humanin peptide (HN) to retinal epithelial cells (ARPE-19). The MSN core, designed with a pore size of 2.8 nm, ensures a high HN loading capacity 64.4% (w/w). We fine-tuned the external surface of the MSNs by incorporating 20% Acetyl-L-arginine (Ar) to create a partial positive charge, while 80% conjugated thioketal (TK) methoxy polyethylene glycol (mPEG) act as ROS gatekeeper. Ex vivo experiments using bovine eyes revealed the immobilization of Ar-MSNs-TK-PEG (mean zeta potential: 2 mV) in the negatively charged vitreous. However, oxidative stress reversed the surface charge to −25 mV by mPEG loss, facilitating the diffusion of the nanoparticles impeded with HN. In vitro studies showed that ARPE-19 cells effectively internalize HN-loaded Ar-MSNs-TK, subsequently releasing the peptide, which offered protection against oxidative stress-induced apoptosis, as evidenced by reduced TUNEL and caspase3 activation. The inhibition of retinal neovascularization was further validated in an in vivo oxygen-induced retinopathy (OIR) mouse model.