Caspase-3 Activity Research Articles

Reduced GATA1 levels are associated with ineffective erythropoiesis in sickle cell anemia.

Ineffective erythropoiesis (IE) is defined as the abnormal differentiation and excessive destruction of erythroblasts in the marrow, accompanied by an expanded progenitor compartment and relative reduction in the production of reticulocytes. It is a defining feature of many types of anemia, including beta-thalassemia. GATA1 is an essential transcription factor for erythroid differentiation, known to be implicated in hematological conditions presenting with IE, including beta-thalassemia and congenital dyserythropoietic anemia. However, little is known about the role of GATA1 in the erythropoietic defects recently described in sickle cell anemia (SCA). In the present study, we performed a detailed characterization of the role of GATA1 and ineffective erythropoiesis in SCA using both invitro and in-vivo assay systems. We demonstrate a significant decrease in GATA1 protein levels during SCA erythropoiesis and a concomitant increase in oxidative stress. Furthermore, we found that an increase in the activity of the inflammatory caspase, caspase 1, was driving the decrease in GATA1 levels during SCA erythropoiesis and that, upon inhibition of caspase 1 activity, SCA erythropoiesis was rescued and GATA1 levels partially restored. Our study further elucidates the defect in erythropoiesis in SCA, and may therefore help in the development of novel approaches to normalise the bone marrow niche prior to stem cell transplantation, or facilitate the production of healthy stem cells for gene therapy.

Upregulation of caspase-3, oxidative stress, neurobehavioural and histological alterations in mercury chloride-exposed rats: role of aqueous Allium sativum bulb extract.

Mercury is a highly toxic metal that causes a variety of neurological disorders through oxidative stress. Allium sativum, a cooking spice in diverse cultures around the world, has a long history of medicinal use due to its rich antioxidant constituents. This study was designed to evaluate the protective activity of aqueous Allium sativum bulb extract (ASBE) on mercuric chloride (HgCl2)-induced neurotoxicity. Forty Wistar rats were randomly divided into five groups namely I (control), II (HgCl2; 4mg/kg), III (250mg/kg of ASBE and 4mg/kg of HgCl2), IV (500mg/kg of ASBE and 4mg/kg of HgCl2) and V (500mg/kg of Vitamin E and 4mg/kg of HgCl2). At the end of the administration, neurobehavioural, antioxidant enzymes, lipid peroxidation and apoptotic activities as well as the histology of the cerebrum, cerebellum and hippocampus were assessed. Body and brain weights, locomotion, exploration, cognition, memory and antioxidant enzymes were significantly impaired (p < 0.05) in HgCl2-exposed rats following comparison to control. Lipid peroxidation, mercury concentration and caspase-3 activity were significantly upregulated (p < 0.05) in HgCl2-exposed rats following comparison to control. In addition, significant alterations to the histology of the cerebrum, cerebellum and hippocampus were observed in the HgCl2-exposed rats. Conversely, the adverse effects induced by HgCl2 were significantly attenuated (p < 0.05) following ASBE and Vitamin E pretreatment. Taken together, these results suggest that ABSE exerts its neuroprotective activity through its potent antioxidant and anti-apoptotic properties.

Protective effect of ascorbic acid against renal injury induced by 3-chloropropane-1,2-diol-dipalmitate in rats.

3-monochloropropane-1,2-diol esters (3-MCPDE) are a group of contaminants which are mainly formed during heat processing of edible oil and fat-based foods. The kidney is the primary target organ for the toxic effects of 3-MCPDE. 3-MCPD-di-palmitate exists in a variety of oils and fats, and is the most common and relatively high proportion of 3-MCPDE. In this study, we investigated the protective effect of ascorbic acid on 3-MCPD-di-palmitate-induced renal injury in rats. Thirty 8-week-old male Sprague-Dawley rats were randomly divided into 5 groups, namely control, 3-MCPD-di-palmitate (240 mg/kg·bw), 3-MCPD-di-palmitate (240 mg/kg·bw) + ascorbic acid (100 mg/kg·bw), 3-MCPD-di-palmitate (240 mg/kg·bw) + ascorbic acid (200 mg/kg·bw) and 3-MCPD-di-palmitate (240 mg/kg·bw) + ascorbic acid (500 mg/kg·bw). These treatments were administered via gavage for a duration of 4 weeks. The effects of ascorbic acid on 3-MCPDE-induced kidney injury in rats were investigated by evaluating the kidney index, renal function (BUN, CRE), renal histopathology, oxidative stress markers (ROS, GSH, MDA, and T-AOC), DNA oxidation marker (8-OHdG), and activities of Caspase 3 and 9. The results showed that the exposure to 3-MCPDE significantly increased the kidney index, BUN and CRE levels, ROS and MDA levels, 8-OHdG levels, and activities of Caspase 3 and 9, while decreasing GSH and T-AOC. The combined treatment with 3-MCPDE and ascorbic acid can effectively restore the aforementioned parameters. The present study concluded that ascorbic acid effectively attenuates the renal apoptosis and oxidative homeostasis induced by 3-MCPDE uptake thereby intervening in renal injury.

Changes in apoptosis in large yellow croaker (Larimichthys crocea) after death: considering the influence of mitochondria and microorganisms

The apoptosis of large yellow croaker stored at 4 °C after death and the effects of five microorganisms extracted from fillets on apoptosis indicators were studied. The results showed that mitochondrial permeability transition pore (MPTP), apoptosis rate, mitochondrial swelling, caspase activity, and cytochrome c (cyt-c) redox state of samples at 0 h were the highest during postmortem storage (p < 0.05). The continuous opening of MPTP and mitochondrial swelling during postmortem storage led to an increase in cyt-c redox status and affected caspase activity. MPTP opening was significantly affected by the inoculation samples with Acinetobacter haemolyticus and Chryseobacterium (p < 0.05). The samples inoculated with Psychrobacter and Pseudomonas had a significant effect on the cyt-c redox state (p < 0.05). The samples inoculated with Pseudomonas and Acinetobacter haemolyticus had a significant effect on caspase activity (p < 0.05). Although this study found that microorganisms can affect apoptosis, it provides a foundation for future in-depth research. However, apoptosis is affected by a variety of signaling molecules, and in order to explore its specific mechanism, more extensive exploration is needed.

Engineering of phosphatidylserine-targeting ROS-responsive polymeric prodrug for the repair of ischemia-reperfusion-induced acute kidney injury

Ischemia-reperfusion-induced acute kidney injury (IR-AKI) commonly occurs in situations such as hemorrhagic shock, kidney transplantation, and cardiovascular surgery. As one of the significant causes of AKI, IR-AKI is characterized by its high incidence and mortality rates. Currently, effective inflammation control is the key for the treatment of IR-AKI. In this study, we developed an ROS-responsive polymeric prodrugs (Zn-D/DTH) which could target the externalized PS of apoptotic cells, and then responsively released HDM (anti-inflammatory peptides) in the presence of intracellular ROS. Zn-D/DTH effectively ameliorated renal function and mitigated pathological alterations such as the loss of the brush border, tubular dilation, and accumulation of cellular debris within the tubular lumens. Furthermore, Zn-D/DTH greatly reduced the generation of pro-inflammatory factors like IL-6, COX-2, and iNOS in renal tissues, suggesting its protective role largely stems from suppression of the inflammatory response. Additional mechanism exploration revealed that Zn-D/DTH markedly decreased the expression levels of TLR4 and MyD88, as well as the phosphorylation of NF-κB in the damaged kidneys. This, in turn, reduced the number of apoptotic tubular cells and the activity of Caspase 9 and Caspase 3 caused by ischemia-reperfusion. Additionally, Zn-D/DTH treatment showed improvement in the long-term renal damage and fibrosis induced by ischemia-reperfusion. The experimental outcomes indicated that Zn-D/DTH attenuated renal ischemia-reperfusion injury and delayed the transition from acute kidney injury to chronic kidney disease by downregulating the TLR4/MyD88/NF-κB signaling pathway and reducing the expression of apoptotic caspases, thereby inhibiting inflammation and reducing cell apoptosis.

Effect of lipoic acid on cisplatin-induced cardiotoxicity and inflammation in cardiomyocytes

Aims: Cisplatin (CPL) is a potent chemotherapeutic agent widely used to treat various cancers. However, CPL causes toxicity in various tissues, including the heart. Lipoic acid (LPA) is a thiol compound with antioxidant, anti-inflammatory, and antiapoptotic properties. Although LPA has been reported to have protective effects in various cardiac diseases, the mechanisms underlying its cardio-protective effects have not been elucidated. This study aimed to establish a CPL-induced cardiotoxicity model in the H9c2 cardiomyocyte cell line, to understand the mechanisms underlying this cardiotoxicity, and to investigate the effect of LPA on cardiotoxicity. Methods: H9c2 cardiomyocyte cells as control (CNT), CPL (40 µm), and LPA-1 (300 µm LPA, and 40 µm CPL), LPA-2 (500 µm LPA, and 40 µm CPL) in combination along with CIS were used. In the analyses made, glutathione (GSH) and glutathione peroxidase (GSHPx) enzyme activity, lipid peroxidation [malondialdehyde, (MDA)] levels, inflammation markers interleukin (IL) -1ß, IL-6, and tumor necrosis factor (TNF) -? levels, total oxidant/antioxidant (TOS and TAS) status levels, reactive oxygen species (ROS) and caspase activity (Casp 3 and 9) in the cells were determined. Results: CIS treatment caused cardiomyocyte cell toxicity and increased Casp 3, Casp 9, ROS, IL-1ß, IL-6, TNF-?, TOS, and MDA levels while decreasing GSHPx, GSH, and TAS levels. Increased inflammation and impaired oxidant/antioxidant balance in cardiomyocyte cells after CPL treatment were regulated by LPA treatment. Conclusion: LPA treatment was found to have a protective effect against CPL-induced cardiotoxicity in cardiomyocyte cells.

Verbascum gimgimense an Endemic Turkish Plant: Evaluation of In Vitro Anticancer, Antioxidant, Enzyme Inhibitory Activities, and Phytochemical Profile.

The Verbascum genus has gained significant attention in the pharmaceutical field, particularly in recent years, due to its valuable medicinal properties, which are well-recognized in complementary and alternative medicine. Certain species within this genus contain essential compounds and exhibit a wide range of therapeutic activities. In this study, the ethanolic extract of Verbascum gimgimense (VG) was analyzed for its cytotoxic, apoptotic, antioxidant, and enzyme inhibitory properties, as well as its phenolic and lipophilic compounds. The phenolic compounds in the extract were identified using Exactive Plus Orbitrap HPLC-HRMS, while the lipophilic components were characterized by GC-MS analysis. The Neutral Red Uptake (NRU) cell viability assay and colony formation assay were performed to assess the antiproliferative and anti-colony survival effects of VG on the A549 human lung adenocarcinoma cell line. Additionally, a wound healing assay measured cell migration, and the apoptotic process was evaluated using Caspase-3 ELISA and acridine orange/ethidium bromide staining. Protein expression levels were determined by western blot analysis. DPPH, ABTS FRAP, and CUPRAC assays were used to determine free radical scavenging, reducing power, and metal chelating activities, respectively. VG was rich in dominant phenolic components, including benzoic acid (6.809 mg/g extract), phloretic acid (1.279 mg/g extract), luteolin 7-rutinoside (2.799 mg/g extract), luteoloside (3.300 mg/g extract), kuromanine (3.456 mg/g extract), and rutin hydrate (2.015 mg/g extract). Major fatty acids identified in VG included palmitic acid (17.3%), stearic acid (2.99%), linoleic acid (9.44%), and α-linolenic acid (26.48%). VG treatment significantly reduced colony formation ability, decreased wound closure, and increased both apoptotic cell count and caspase-3 activity compared to the control group. Protein levels of c-PARP, p53, and p21 were substantially elevated compared to controls. In addition to its strong free radical scavenging, reducing power and metal chelating activity, VG exhibited strong inhibitory effects on α-amylase, α-glucosidase, AChE, BChE, and tyrosinase. Our study demonstrates that VG possesses antiproliferative, apoptotic, antioxidant, and enzyme-inhibitory properties. V. gimgimense emerges as a promising natural antioxidant source with potentially significant regulatory effects on key enzymes and proteins, which could contribute to managing various human diseases and inspire the development of novel therapeutic strategies.

Silver-Doped Zinc Ferrite Nanoparticles: Trigger ROS-Dependent Apoptosis and Inhibit Migration in MCF-7 Breast Cancer Cells

This study describes the fabrication of nanostructured silver-doped zinc ferrite nanoparticles (AgxZn1-xFe2O4, where x = 0.0 and 0.05) using a chemical co-precipitation method. Field-emission scanning electron microscopy (FE-SEM) analysis was performed to investigate the surface characteristics and particle size of the pure and Ag-doped nanoparticles. X-ray diffraction (XRD) patterns confirmed the formation of a single-phase cubic structure for the zinc ferrite nanoparticles. The anti-cancer potential of the nanoparticles was evaluated using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) against human breast cancer cells (MCF-7). The results showed that (AgxZn1-xFe2O4, where x = 0.05) nanoparticles with x = 0.05 exhibited significant cytotoxicity. At a concentration of 100 µg/mL, these nanoparticles reduced cell viability to 26.27%, with a calculated LC50 value (concentration lethal to 50% of cells) of 41.30 µg/mL after 24 hours of treatment. Notably, the LC50 value for (AgxZn1-xFe2O4, where x = 0.05) nanoparticles in normal L929 fibroblast cells was over four times higher compared to MCF-7 cancer cells, indicating a degree of selectivity for cancer cells. Further investigation revealed that (AgxZn1-xFe2O4, where x = 0.05) treatment significantly increased the generation of reactive oxygen species (ROS) within the cancer cells. This was confirmed by fluorescence intensity measurements, which showed a substantial increase from 1260.61 (control) to 15600 for cells treated with (AgxZn1-xFe2O4, where x = 0.05) nanoparticles. This included flow cytometry analysis of apoptosis (programmed cell death), migration assays to evaluate metastasis potential, measurement of antioxidant enzyme activity (SOD and catalase) to understand the impact on the cellular antioxidant system, indirect ELISA to detect caspase activity (a marker of apoptosis), cell cycle analysis, and finally, real-time PCR analysis to determine the mRNA expression of p53, a tumor suppressor gene. The apoptosis assay confirmed a significant increase in apoptotic cells upon treatment with (AgxZn1-xFe2O4, where x = 0.05) nanoparticles. This activity is likely mediated by the generation of ROS and subsequent oxidative stress within the cancer cells. These findings highlight the potential of these nanoparticles as a promising therapeutic strategy for cancer treatment.

METTL14 Promotes Lipopolysaccharide-Induced Myocardial Damage via m6A-Dependent Stabilization of TRPM7 mRNA.

Sepsis-induced myocardial injury (SIMI) is a vital pathological component of severe sepsis and septic shock. As a prevalent internal mRNA modification in eukaryotic cells, N6-methyladenosine (m6A) modification is implicated in sepsis and immune disorders. Methyltransferase-like 14 (METTL14), a core subunit of the methyltransferase complex that catalyzes messenger RNA m6A modification, is involved in the regulation of human cardiomyocyte cell line (AC16) injury. This study aimed to explore the role and mechanism of METTL14 in lipopolysaccharide (LPS) -induced myocardial injury.Cell viability and apoptosis were analyzed via 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and TdT-mediated dUTP nick-end labeling (TUNEL) assay. The Tumor necrosis factor alpha (TNF-α) and Interleukin-1beta (IL-1β) levels were analyzed via Enzyme linked immunosorbent assay (ELISA). Caspase-3 activity, reactive oxygen species activity, malondialdehyde level, and glutathione level were assessed using special assay kits. The levels of transient receptor potential melastatin 7 (TRPM7) and METTL14 mRNA were determined via Real-time quantitative polymerase chain reaction (RT-qPCR). Meanwhile, the protein levels of TRPM7, METTL14, phospho-p65 (p-p65), total p65 (p65), p-IκBα, and total IκBα (IκBα) were examined via western blot assay.LPS treatment repressed AC16 cell viability and induced cell apoptosis, inflammatory response, oxidative stress, and ferroptosis in vitro. METTL14 and TRPM7 were upregulated in LPS-treated AC16 cells. At the molecular level, METTL14 could increase the stability of TRPM7 mRNA via m6A methylation. Moreover, METTL14 deficiency could abolish LPS-triggered AC16 cell injury and ferroptosis via TRPM7 regulation.METTL14 knockdown reversed LPS-caused myocardial cell damage mainly by regulating the stability of TRPM7 mRNA, providing a novel therapeutic target for septic cardiomyopathy treatment.

Preventive effects of melatonin and amifostine on irradiated rats with experimental periodontitis

BackgroundThe aim of this study was to investigate the preventive effects of amifostine and melatonin oxidatively, biochemically and histomorphometrically in rats with radiotherapy-induced experimental periodontitis.Methods40 female Sprague-Dawley rats were divided into 5 groups: Control, experimental periodontitis (Ep), Ep + radiotherapy (Ep + Rt), Ep + Rt + amifostine (Ep + Rt + Ami), Ep + Rt + melatonin (Ep + Rt + Mel). The day after induction of periodontitis by ligature, a single dose of 5 Gy radiotherapy was administered. On the same day, treatments with amifostine (200 mg/kg) for 3 days and melatonin (10 mg/kg) for 15 days were started. By after 23 days of experiment, periodontal bone loss was measured by histomorphometry. RANKL, OPG and Caspase-3 activities were analyzed immunohistochemically and inflammatory cytokine (IL-1β, IL-10, IL-6, TNF-α) levels and oxidative stress (TOS/TAS) were analyzed biochemically in tissue homogenates.ResultsIt was observed that there was a significant difference in many biochemical parameters and oxidative stress levels between the control group and Ep + Rt (p < 0.05). Alveolar bone destruction in the melatonin prophylaxis group was observed to be close to control (p > 0.05). Melatonin significantly improved biochemical, histochemical, apoptotic and bone loss levels in irradiated experimental periodontitis rats (p < 0.05). When comparing the two drug groups (Ep + Rt + Ami and Ep + Rt + Mel), no statistically significant difference was found at any parameter level (p > 0.05).ConclusionBoth melatonin and amifostine can significantly limit RT-induced periodontal bone loss by suppressing inflammatory stress, apoptotic mechanisms, and RANKL-related osteoclastic activity. Given the limited side effects of melatonin, it may be an alternative to amifostine.

In vitro assessment of the role of endoplasmic reticulum stress in sunitinib-induced liver and kidney toxicity

Sunitinib, a multi-targeted tyrosine kinase inhibitor, is prescribed for the treatment of metastatic gastrointestinal stromal tumors, advanced metastatic renal cell carcinoma, and pancreatic neuroendocrine tumors. Hepatotoxicity and nephrotoxicity are significant adverse effects of sunitinib administration; however, there is limited information regarding the molecular mechanisms of these adverse effects. The aim of the present study was to elucidate the role of endoplasmic reticulum stress in hepatotoxicity and nephrotoxicity induced by sunitinib. In addition to endoplasmic reticulum stress, oxidative stress and mitochondrial membrane potential were evaluated to investigate the molecular mechanism more comprehensively. Findings revealed that sunitinib exposure significantly increased the reactive oxygen species levels and decreased the Nrf2 gene expression and GSH/GSSG ratio, suggesting oxidative stress induction in normal hepatocyte (AML12) and normal kidney (HK-2) cell lines. Endoplasmic reticulum stress markers, including ATF4, CHOP, IRE1α, XBP1s and ATF6 mRNA expressions, were upregulated in AML12 cells. Furthermore, enhanced intracellular calcium levels also indicate endoplasmic reticulum stress in hepatocytes. In contrast, sunitinib exposure did not alter endoplasmic reticulum-related gene expression levels and intracellular calcium levels in HK-2 cells. In terms of mitochondrial membrane potential and caspase-3 activity, sunitinib induced mitochondrial membrane damage and increased caspase-3 activation not only in AML12 cells but also in HK-2 cells. The research findings indicate that sunitinib may induce cytotoxic effects in hepatocytes through mechanisms involving oxidative stress, endoplasmic reticulum stress, and mitochondrial damage. However, in the kidney, the toxicity mechanism is different from that of liver, and the endoplasmic reticulum stress does not seem to be involved in this mechanism.

The protumorigenic enzyme GPAT2 inhibits arachidonic acid-triggered apoptosis in breast cancer

BackgroundCancer is a significant health challenge and the leading cause of mortality globally. Tumor cells use multiple mechanisms to acquire their distinctive capacity for uncontrolled proliferation, one of which is the evasion of apoptosis. It has been shown that in breast, colon, and liver cancer, evasion of apoptosis is associated with the overexpression of enzymes that metabolize arachidonic acid (AA) because free AA is a strong inducer of apoptosis. Glycerol−3-phosphate acyltransferase 2 (GPAT2) is a key enzyme in AA metabolism and is highly expressed in breast and colon cancer, where it promotes the development of essential tumor features.MethodsIn this work, a model of GPAT2 silencing in the human breast cancer-derived cell line MDA-MB-231 was used, and the cells were exposed to exogenous AA. The role of GPAT2 in AA-induced cell death was studied using MTT and TUNEL assays and measurements of caspase activity. The underlying molecular mechanism of cell death was assessed by qRT‒PCR.ResultsThe results showed that AA reduced cell viability only in GPAT2-silenced cells, and that this cell death was a consequence of an apoptotic process involving BNIP3 overexpression. Additionally, it was demonstrated that GPAT2 silencing triggered a compensatory mechanism by overexpressing other genes involved in AA utilization for eicosanoid biosynthesis.ConclusionsWe concluded that GPAT2 expression is necessary to prevent AA-induced apoptotic cell death in MDA-MB-231 cells and that the overexpression of other AA-metabolizing genes is not sufficient to compensate for the lack of GPAT2 and prevent apoptosis.

Detoxification of paraoxon-ethyl by Lysiphyllum strychnifolium extracts in undifferentiated human neuroblastoma SH-SY5Y cells

Context Lysiphyllum strychnifolium (Craib) A. Schmitz. (Fabaceae) is a Thai traditional medicine used to remove food and alcohol toxins from the body. Objective This study investigates the molecular mechanism of L. strychnifolium extracts against paraoxon-ethyl-induced apoptosis in SH-SY5Y cells. Materials and methods The ethanol and water extracts of leaves and stems of L. strychnifolium were prepared at various concentrations (0–100 μg/mL) and co-treated to the cells with 0.375 mM paraoxon-ethyl for 24 and 48 h. Cell viability was performed using the PrestoBlue assay. ROS and caspase activity were detected using 2′,7′-dichlorodihydrofluorecein diacetate and caspase-Glo® 3/7, 8, and 9 assay kits. Apoptotic and ER stress-related gene expression were determined by real-time PCR, and nuclear and mitochondrial morphology were observed using Hoechst 33342 and MitoTracker® Deep Red FM staining. Results The most effective concentrations of each extract against paraoxon-ethyl-induced cell death were 25 μg/mL of leaf ethanol, 12.5 μg/mL of stem ethanol, 100 μg/mL of leaf water, and 25 μg/mL of stem water extracts. The leaf ethanol extract was the most effective at detoxifying, while stem extracts were highly toxic in high doses. The detoxifying L. strychnifolium extracts against paraoxon-ethyl-induced oxidative stress decreased p53, BiP/GRP78, and CHOP gene expression and minimized caspase 9 and caspase 3, protecting cells from apoptosis. The extracts could also restore mitochondrial membrane potential and reduce the swollen globule mitochondrial shape. Discussion and conclusion These findings could potentially protect neuron cells from neurodegenerative issues due to oxidative damage, apoptosis, and other potential consequences.

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 affects 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. Accordingly, we found that multiple effects of emc mutations on cell growth and on eye development were all caused by 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. Growth inhibition of emc mutant clones in wing imaginal discs also depended on caspases. 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.

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 affects 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. Accordingly, we found that multiple effects of emc mutations on cell growth and on eye development were all caused by 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. Growth inhibition of emc mutant clones in wing imaginal discs also depended on caspases. 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.

Treatment of Melanoma Cells with Chloroquine and Everolimus Activates the Apoptosis Process and Alters Lipid Redistribution.

The balance between apoptosis and autophagy plays a key role in cancer biology and treatment strategies. The aim of this study was to assess the effect of the mTOR kinase inhibitor everolimus and chloroquine on the regulation of proliferation, caspase-3 activation, and apoptosis in melanoma cells. We studied the activity of caspase-3 and the levels of caspase-3 and -9 using the Western blot technique. Cellular apoptosis was examined using a DNA fragmentation assay, and changes in the cell nucleus and cytoskeleton were examined using fluorescence microscopy DAPI, OA/IP. We also studied the rearrangement of lipid structures using fluorescent dyes: Nile Red and Nile Blue. A low nanomolar concentration of the mTOR kinase inhibitor everolimus in combination with chloroquine activated the apoptosis process and decreased cell proliferation. These changes were accompanied by an obvious change in cell morphology and rearrangement of lipid structures. Alterations in lipid redistribution accompanying the process of apoptosis and autophagy are among the first to occur in the cell and can be easily monitored in in vitro studies. The combination of mTOR inhibitors and chloroquine represents a promising area of research in cancer therapy. It has the potential to enhance treatment efficacy through complementary mechanisms.

Ally or traitor: the dual role of p62 in caspase-2 regulation

Caspase-2 is a unique and conserved cysteine protease that is involved in several cellular processes, including different forms of cell death, maintenance of genomic stability, and the response to reactive oxygen species. Despite advances in caspase-2 research in recent years, the mechanisms underlying its activation remain largely unclear. Although caspase-2 is activated in the PIDDosome complex, its processing could occur even in the absence of PIDD1 and/or RAIDD, suggesting the existence of an alternative platform for caspase-2 activation. Here, we show that caspase-2 undergoes ubiquitination and interacts with scaffolding protein p62/sequestosome-1 (SQSTM1) under normal conditions and in response to DNA damage. p62 promotes proteasomal but not autophagic caspase-2 degradation as well as its dimerization and activation that triggers the caspase cascade and, subsequently, cell death. Inhibition of p62 expression attenuates cisplatin-induced caspase-2 processing and apoptosis. Notably, the ZZ domain of p62 is critical for caspase-2 binding, whereas the UBA domain is seemingly required to stabilize the p62–caspase-2 complex. Thus, we have uncovered the dual role of p62 in regulating caspase-2 activity: it can foster the degradation of caspase-2 in the proteasome or facilitate its activation by acting as a scaffold platform.

Transfection of 12/15-lipoxygenase effectively alleviates inflammatory responses during experimental acute pancreatitis

BACKGROUND Acute pancreatitis (AP), the initially triggered inflammatory process in the pancreas, can be life-threatening. It has been reported that 15-lipoxygenase may promote the removal of damaged intracellular components, maintain intracellular homeostasis, and promote apoptosis by upregulating the activity of caspases. Despite an increased understanding of the lipoxygenase pathway in inflammation and immune diseases, the role of the Alox15 gene product in modulating the inflammatory changes during AP is not well defined. AIM To investigate the effect of Alox15 expression in cerulein-induced AP in rats. METHODS Model rats were transfected with Alox15 by injecting a recombinant lentivirus vector encoding Alox15 into the left gastric artery before inducing AP. The expression of Alox15 was then assessed at the mRNA and protein levels. RESULTS Our in vivo results showed that serum amylase activity and pancreatic tissue water content were significantly reduced in Alox15 -transfected rats. Further, the mRNA expression levels of tumor necrosis factor alpha, interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein-1, as well as the protein expression of nuclear factor kappa B in pancreatic tissue were reduced. Additionally, we observed an upregulation of cleaved caspase-3 that implies an induction of apoptosis in pancreatic cells. The transfection of Alox15 resulted in a lower number of autophagic vacuoles in AP. CONCLUSION Our findings demonstrate a regulatory role of Alox15 in apoptosis and autophagy, making it a potential therapeutic target for AP.

Caspase-3 Activity and Autophagy Expression in the Development of Neuronal Resistance to Glutamate Toxicity

Two periods of autophagy activation, differently significant for the development of resistance, were demonstrated in the model of neuronal resistance to the toxic effect of glutamate (deprivation of trophic factors). The autophagy inhibitor 3-methyladenine (3-MA) at a concentration of 1.25 mM significantly suppresses resistance development, but only if applied immediately after deprivation of trophic factors. Inhibition of autophagy with 3-MA immediately during deprivation does not affect resistance production. In addition, activation of autophagy is responsible for the decrease in caspase-3 activity, although the mechanism of this process remains unclear. We hypothesize that resistance production in neurons is mediated by a decrease in caspase-3 activity as a result of autophagy activation.

Adipocyte Apoptosis Following a Novel Method for Double Chin Reduction: A Pilot Human Histology Study.

Submental fullness is perceived as unattractive by both men and women. The noninvasive simultaneous delivery of HIFES and synchronized radiofrequency+ (Sync RF+) technologies aims to address the submental fullness by concurrently targeting the skin, adipose tissue, and weakened anterior belly of the digastric muscle, the three contributing layers to the double chin appearance. This study aims to investigate the histological changes to adipose tissue related to cell morphology, caspase-7, and Bcl-2 levels to detect adipocyte apoptosis following the HIFES and Sync RF+ treatment on human subjects. The active group (n = 6) received single 20-min treatment on the submental area, while the control group (n = 2) did not receive any treatment. Biopsies of subcutaneous fat tissue were obtained at baseline, and 24 h and 7 days posttreatment. The specimens were histologically and immunohistochemically analyzed for changes in morphology, caspase-7, and Bcl-2 levels. Observed caspase-7 levels increased by 511% at 24-h posttreatment, and 101% at 7 days (p < 0.0001), while the Bcl-2 levels decreased by 89% at 24 h and 24% at 7 days posttreatment (p < 0.0001). The control group had no statistically significant relative changes in the activity of caspase-7. Posttreatment adipocytes were shrunken in size, and shapes lost their uniformity compared to baseline. Five of six subjects reported the treatment as being comfortable. No adverse events were observed during the study. The results of this human histology study indicate that noninvasive HIFES and Sync RF+ technologies have a favorable safety profile for submental fat reduction through the induction of adipocyte apoptosis. ClinicalTrials.gov identifier: NCT06282172.