ABSTRACT
Aim: The main goal was to create two new groups of indole derivatives, hydrazine-1-carbothioamide (4a and 4b) and oxadiazole (5, and 6a-e) that target EGFR (4a, 4b, 5) or VEGFR-2 (6a-e). Materials & methods: The new derivatives were characterized using various spectroscopic techniques. Docking studies were used to investigate the binding patterns to EGFR/VEGFR-2, and the anti-proliferative properties were tested in vitro. Results: Compounds 4a (targeting EGFR) and 6c (targeting VEGFR-2) were the most effective cytotoxic agents, arresting cancer cells in the G2/M phase and inducing the extrinsic apoptosis pathway. Conclusion: The results of this study show that compounds 4a and 6c are promising cytotoxic compounds that inhibit the tyrosine kinase activity of EGFR and VEGFR-2, respectively.
[Box: see text].
Subject(s)
Antineoplastic Agents , Cell Proliferation , ErbB Receptors , Indoles , Vascular Endothelial Growth Factor Receptor-2 , Humans , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Indoles/chemistry , Indoles/pharmacology , Molecular Docking Simulation , Molecular Structure , Structure-Activity Relationship , /pharmacology , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are common retinal diseases responsible for most blindness in working-age and elderly populations. Oxidative stress and mitochondrial dysfunction play roles in these pathogenesis, and new therapies counteracting these contributors could be of great interest. Some molecules, like coenzyme Q10 (CoQ10), are considered beneficial to maintain mitochondrial homeostasis and contribute to the prevention of cellular apoptosis. We investigated the impact of adding CoQ10 (Q) to a nutritional antioxidant complex (Nutrof Total®; N) on the mitochondrial status and apoptosis in an in vitro hydrogen peroxide (H2O2)-induced oxidative stress model in human retinal pigment epithelium (RPE) cells. H2O2 significantly increased 8-OHdG levels (p < 0.05), caspase-3 (p < 0.0001) and TUNEL intensity (p < 0.01), and RANTES (p < 0.05), caspase-1 (p < 0.05), superoxide (p < 0.05), and DRP-1 (p < 0.05) levels, and also decreased IL1ß, SOD2, and CAT gene expression (p < 0.05) vs. control. Remarkably, Q showed a significant recovery in IL1ß gene expression, TUNEL, TNFα, caspase-1, and JC-1 (p < 0.05) vs. H2O2, and NQ showed a synergist effect in caspase-3 (p < 0.01), TUNEL (p < 0.0001), mtDNA, and DRP-1 (p < 0.05). Our results showed that CoQ10 supplementation is effective in restoring/preventing apoptosis and mitochondrial stress-related damage, suggesting that it could be a valid strategy in degenerative processes such as AMD or DR.
Subject(s)
Apoptosis , Hydrogen Peroxide , Oxidative Stress , Retinal Pigment Epithelium , Ubiquinone , Humans , Ubiquinone/analogs & derivatives , Ubiquinone/pharmacology , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/drug effects , Oxidative Stress/drug effects , Apoptosis/drug effects , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , Mitochondria/metabolism , Mitochondria/drug effects , Antioxidants/pharmacology , Epithelial Cells/metabolism , Epithelial Cells/drug effects , Cell Line , Dietary SupplementsABSTRACT
BACKGROUND: The cysteine-aspartic acid protease caspase-3 is recognized as the main executioner of apoptosis in cells responding to specific extrinsic and intrinsic stimuli. Caspase-3 represents an interesting biomarker to evaluate treatment response, as many cancer therapies exert their effect by inducing tumour cell death. Previously developed caspase-3 PET tracers were unable to reach routine clinical use due to low tumour uptake or lack of target selectivity, which are two important requirements for effective treatment response evaluation in cancer patients. Therefore, the goal of this study was to develop and preclinically evaluate novel caspase-3-selective activity-based probes (ABPs) for apoptosis imaging. RESULTS: A library of caspase-3-selective ABPs was developed for tumour apoptosis detection. In a first attempt, the inhibitor Ac-DW3-KE (Ac-3Pal-Asp-ßhLeu-Phe-Asp-KE) was 18F-labelled on the N-terminus to generate a radiotracer that was incapable of adequately detecting an increase in apoptosis in vivo. The inability to effectively detect active caspase-3 in vivo was likely attributable to slow binding, as demonstrated with in vitro inhibition kinetics. Hence, a second generation of caspase-3 selective ABPs was developed based on the Ac-ATS010-KE (Ac-3Pal-Asp-Phe(F5)-Phe-Asp-KE) with greatly improved binding kinetics over Ac-DW3-KE. Our probes based on Ac-ATS010-KE were made by modifying the N-terminus with 6 different linkers. All the linker modifications had limited effect on the binding kinetics, target selectivity, and pharmacokinetic profile in healthy mice. In an in vitro apoptosis model, the least hydrophilic tracer [18F]MICA-316 showed an increased uptake in apoptotic cells in comparison to the control group. Finally, [18F]MICA-316 was tested in an in vivo colorectal cancer model, where it showed a limited tumour uptake and was unable to discriminate treated tumours from the untreated group, despite demonstrating that the radiotracer was able to bind caspase-3 in complex mixtures in vitro. In contrast, the phosphatidylethanolamine (PE)-binding radiotracer [99mTc]Tc-duramycin was able to recognize the increased cell death in the disease model, making it the best performing treatment response assessment tracer developed thus far. CONCLUSIONS: In conclusion, a novel library of caspase-3-binding PET tracers retaining similar binding kinetics as the original inhibitor was developed. The most promising tracer, [18F]MICA-316, showed an increase uptake in an in vitro apoptosis model and was able to selectively bind caspase-3 in apoptotic tumour cells. In order to distinguish therapy-responsive from non-responsive tumours, the next generation of caspase-3-selective ABPs will be developed with higher tumour accumulation and in vivo stability.
ABSTRACT
The successful treatment of Alzheimer's disease (AD) is still a big challenge. Rivastigmine is one of the most used drugs for the treatment of AD. The short half-life, lower bioavailability, and less concentration of the drug in the brain after oral delivery are considered the main drawbacks of rivastigmine. To improve these drawbacks, nanostructure-mediated drug delivery has gained more attention. This study investigates the effect of rivastigmine-loaded in optimized chitosan nano-particles (RS-CSNPs) as polymeric nano-carriers by different administration routes (oral and intranasal) on aluminum chloride (AlCl3)-induced Alzheimer-like disease in rat. The model was established by giving rats 100 mg/kg/b.wt of AlCl3 orally for 3 months. Then the experimental rats were treated with RS-CSNPs either orally or intranasally for 75 days. Histopathology, immunohistochemistry of Tau expression in brain tissue, and gene expression of Caspase-3, NF-κB, and Nrf-2 were carried out. The therapeutic agents used decreased the alterations observed in AlCl3 group with improvement in the neuronal viability. In addition to low expression of tau protein, down-regulation of caspase-3 and NF-κB genes and up-regulation of Nrf-2. RS-CSNPs alleviated the progression of AD presumably via blocking the inflammatory cascade and decreasing the oxidative stress process. The intranasal route is superior to the oral one and promising in AD management.
ABSTRACT
Background: Organ ischemia-reperfusion (IR) is a common clinical condition associated with various situations such as trauma surgery, organ transplantation, and myocardial ischemia. Current therapeutic methods for IR injury have limitations, and nanotechnology, particularly zinc oxide nanoparticles (ZnO NPs), offers new approaches for disease diagnosis and treatment. In this study, we investigated the protective and anti-apoptotic effects of ZnO NPs in liver ischemia-reperfusion (IR) injury in rats. Methods: Forty-eight male rats were divided into six groups: sham, ZnO5, ZnO10, ischemia-reperfusion (IR), IR+ZnO5, and IR+ZnO10. The protective effect of ZnO NPs was evaluated by liver enzymes (AST, ALT, Bilirubin, ALP), biochemical (TAC, TNF-α, and MDA), molecular examinations (Bcl2, BAX), and histopathological evaluations (H&E, TUNEL). Results: Pre-treatment with ZnO5 and ZnO10 improved hepatic function in IR liver injury, attenuated the levels of oxidants (P = 0.03) and inflammatory mediators, and reduced apoptosis (P = 0). ZnO10 was found to have a greater effect on ischemic reperfusion injury than ZnO5 did. Histopathological examination also showed a dose-dependent decrease in alterations in the IR+ZnO5 and IR+ZnO10 groups. Conclusion: Administration of ZnO5 and ZnO10 improved liver function after IR. The findings of this study suggest that ZnO NPs have a protective effect against oxidative stress and apoptosis in liver ischemia-reperfusion injury in rats. These results may have important implications for developing advanced methods in ischemia-reperfusion treatment.
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In this work, we report on an electrochemical method for the signal-on detection of caspase-3 and the evaluation of apoptosis based on the biotinylation reaction and the signal amplification of methylene blue (MB)-loaded metal-organic frameworks (MOFs). Zr-based UiO-66-NH2 MOFs were used as the nanocarriers to load electroactive MB molecules. Recombinant hexahistidine (His6)-tagged streptavidin (rSA) was attached to the MOFs through the coordination interaction between the His6 tag in rSA and the metal ions on the surface of the MOFs. The acetylated peptide substrate Ac-GDEVDGGGPPPPC was immobilized on the gold electrode. In the presence of caspase-3, the peptide was specifically cleaved, leading to the release of the Ac-GDEVD sequence. A N-terminal amine group was generated and then biotinylated in the presence of biotin-NHS. Based on the strong interaction between rSA and biotin, rSA@MOF@MB was captured by the biotinylated peptide-modified electrode, producing a significantly amplified electrochemical signal. Caspase-3 was sensitively determined with a linear range from 0.1 to 25 pg/mL and a limit of detection down to 0.04 pg/mL. Further, the active caspase-3 in apoptosis inducer-treated HeLa cells was further quantified by this method. The proposed signal-on biosensor is compatible with the complex biological samples and shows great potential for apoptosis-related diagnosis and the screening of caspase-targeting drugs.
Subject(s)
Biosensing Techniques , Caspase 3 , Metal-Organic Frameworks , Methylene Blue , Metal-Organic Frameworks/chemistry , Methylene Blue/chemistry , Humans , Caspase 3/metabolism , HeLa Cells , Biosensing Techniques/methods , Electrochemical Techniques/methods , Apoptosis , Streptavidin/chemistry , Biotinylation , Electrodes , Limit of Detection , Zirconium/chemistry , Phthalic AcidsABSTRACT
Objectives: The purpose of this study was to determine the correlation between microscopic degeneration in the long head of the biceps tendon (LHBT) and the apoptotic process. Methods: This study included 26 consecutive patients who had undergone arthroscopic biceps tenodesis or tenotomy for symptomatic LHBT with or without concomitant rotator cuff tears (RCTs). Histological examination of the specimens under a light microscope was conducted after staining with hematoxylin, eosin, and the Alcian blue. Histopathological changes were assessed using the original Bonar score and the modified Bonar score and then correlated with the expression of the subsequent apoptosis markers: activated caspase-3 (casp3), tumor protein p53 (p53), and B-cell lymphoma 2 (BCL-2). Results: The mean original Bonar score was 8.65 (range 5-11), while the modified Bonar score was 7.61. There was no correlation between the original Bonar score and the age of the patients, but a positive correlation was found between the modified Bonar score and the age of the patients (p = 0.0022). There was no correlation between the age of patients and the expression indexes of BCL-2 and casp3. However, the expression of the p53 index showed a positive correlation with patient aging (p = 0.0441). Furthermore, there was no correlation observed between the expression of apoptotic indexes and both the original and modified Bonar scale. Conclusions: In LHB tendinopathy, the expression of apoptosis does not seem to directly correlate with the extent of degeneration, particularly in the late stages of tendinopathy. However, the transformations observed in collagen and ground substance were significantly associated with age, as well as tendinous tissue degeneration quantified according to modified Bonar score. The age of patients was also linked with the expression of the p53 index, as an increased apoptosis in the studied population.
ABSTRACT
Despite significant progress in combining cancer immunotherapy with chemotherapy to treat triple negative breast cancer (TNBC), challenges persist due to target depletion and tumor heterogeneity, especially in metastasis. Chemotherapy lacks precise targeting abilities, and targeted therapy is inadequate in addressing the diverse heterogeneity of tumors. To address these challenges, we introduce RGDEVD-DOX as a tumor-specific immunogenic agent, namely TPD1, which targets integrin αvß3 and gets continuously activated by apoptosis. TPD1 facilitates the caspase-3-mediated in situ amplification that results in tumor-specific accumulation of doxorubicin. This local concentration of doxorubicin induces immunogenic cell death and promotes the recruitment of immune cells to the tumor site. Notably, the tumor-targeting capabilities of TPD1 help bypass the systemic immunotoxicity of doxorubicin. Consequently, this alters the tumor microenvironment, converting it into a 'hot' tumor that is more susceptible to immune checkpoint inhibition. We demonstrated the anti-metastatic and anti-cancer efficacy of this treatment using various xenograft and metastatic models. This study underscores the high potential of caspase-3 cleavable peptide-drug conjugates to be used in conjunction with anti-cancer immunotherapies.
ABSTRACT
It has been documented that caspase 3 activity is necessary for skeletal muscle regeneration, but how its activity is regulated is largely unknown. Our previous report shows that intracellular TMEM16A, a calcium activated chloride channel, significantly regulates caspase 3 activity in myoblasts during skeletal muscle development. By using a mouse line with satellite cell (SC)-specific deletion of TMEM16A, we examined the role of TMEM16A in regulating caspase 3 activity in SC (or SC-derived myoblast) as well as skeletal muscle regeneration. The mutant animals displayed apparently impaired regeneration capacity in adult muscle along with enhanced ER stress and elevated caspase 3 activity in Tmem16a-/- SC derived myoblasts. Blockade of either excessive ER stress or caspase 3 activity by small molecules significantly restored the inhibited myogenic differentiation of Tmem16a-/- SCs, indicating that excessive caspase 3 activity resulted from TMEM16A deletion contributes to the impaired muscle regeneration and the upstream regulator of caspase 3 was ER stress. Our results revealed an essential role of TMEM16A in satellite cell mediated skeletal muscle regeneration by ensuring a moderate level of caspase 3 activity.
ABSTRACT
AIM: This study was aimed at investigating the cytotoxic effect of a novel combination of doxorubicin (DOX) and nano-formulation of Santolina chamaecyparissus L. essential oil (SCEO-NANO) on hepatic (HepG2) and colon (HT29) cancer cell lines. METHODS: A nano-emulsion was prepared by high-pressure homogenisation, then analysed by zetasizer and Fourier transform infrared spectroscopy. HepG2 and HT29 cells were used in in vitro tests for apoptosis detection. RESULTS: Formulated droplet size increased in DOX@SCEO-NANO/DOX to 11.54 ± 0.02 with uniform distribution (PDI = 0.13 ± 0.01), when compared with SCEO-NANO (size: 8.91 ± 0.02 nm; PDI = 0.1 ± 0.02). In both cells, DOX@SCEO-NANO/DOX led to a considerable reduction in colony formation. Compared to DOX, apoprotein proteins were overexpressed in HepG2 cells, showing increases of 8.66-fold for caspase-3 and 4.24-fold for the Bax/Bcl-2 ratio. In HT29 cells, ROS-dependent necrosis and apoptosis were seen. Comparing DOX@SCEO-NANO/DOX versus DOX, greater levels of caspase-3 and the Bax/Bcl-2 ratio were observed. CONCLUSION: The DOX@SCEO-NANO/DOX formulation showed potential for targeted eradication of colon adenocarcinoma and hepatocellular carcinoma cells.
ABSTRACT
Coronary artery calcification (CAC) is an early marker for atherosclerosis and is mainly induced by the osteoblast-like phenotype conversion of vascular smooth muscle cells (VSMCs). Recent reports indicate that NOD-like receptor protein 3 (NLRP3)-mediated pyroptosis plays a significant role in the calcification of vascular smooth muscle cells (VSMCs), making it a promising target for treating calcific aortic valve disease (CAC). Ligustrazine, or tetramethylpyrazine (TMP), has been found effective in various cardiovascular and cerebrovascular diseases and is suggested to inhibit NLRP3-mediated pyroptosis. However, the function of TMP in CAC is unknown. Herein, influences of TMP on ß-glycerophosphate (ß-GP)-stimulated VSMCs and OPG-/- mice were explored. Mouse Aortic Vascular Smooth Muscle (MOVAS-1) cells were stimulated by ß-GP with si- caspase-3, si- Gasdermin E (GSDME) or TMP. Increased calcification, reactive oxygen species (ROS) level, Interleukin-1beta (IL-1ß) and Interleukin-18 (IL-18) levels, lactate dehydrogenase (LDH) release, enhanced apoptosis, and activated cysteine-aspartic acid protease-3 (caspase-3)/GSDME signaling were observed in ß-GP-stimulated MOVAS-1 cells, which was sharply alleviated by si-caspase-3, si-GSDME or TMP. Furthermore, the impact of TMP on the ß-GP-induced calcification and injury in MOVAS-1 cells was abolished by raptinal, an activator of caspase-3. Subsequently, OPG-/- mice were dosed with TMP or TMP combined with raptinal. Calcium deposition, increased nodules, elevated IL-1ß and IL-18 levels, upregulated CASP3 and actin alpha 2, smooth muscle (ACTA2), and activated caspase-3/GSDME signaling in OPG-/- mice were markedly alleviated by TMP, which were notably reversed by the co-administration of raptinal. Collectively, TMP mitigated CAC by inhibiting caspase-3/GSDME mediated pyroptosis.
ABSTRACT
BACKGROUND: Several medications, including antihistamines, can alter salivary gland function, causing dry mouth or xerostomia. Antihistamines are commonly used for treating allergic rhinitis. OBJECTIVES: The aim of the present study was to compare and correlate the effects of first-generation vs. second-generation H1-antihistamines on the parotid glands of rats. MATERIAL AND METHODS: Twelve adult male albino rats were used; 4 rats served as a control group (group I) and the remaining rats were divided into 2 groups: group II received promethazine hydrochloride; and group III received cetirizine dihydrochloride for 3 weeks. The parotid salivary glands were dissected, and examined histologically and analyzed histomorphometrically for the acinar area percentage. In addition, mRNA gene expression of iNOS, caspase-3 and α-SMA was assessed using quantitative realtime polymerase chain reaction (qRT-PCR). Finally, all the obtained data was statistically analyzed. RESULTS: Histologically, group I showed the typical architecture of the gland. In group II, degenerative changes were noticed, including acinar degeneration and shrinkage with widened connective tissue septa, intracellular vacuolization, and increased inflammatory cell infiltration. In group III, similar histological features were detected as in group II, but to a lesser extent. Histomorphometric results revealed significant differences in the acinar area percentage between various groups. In addition, qRT-PCR results showed a significant increase in iNOS expression in both groups II and III as compared to group I, caspase-3 gene expression was significantly increased in group II, while in group III, it increased non-significantly. Finally, α-SMA gene expression non-significantly decreased in both groups II and III. A significant positive correlation was observed between caspase-3 and iNOS gene expression, while an inverse correlation was noticed between caspase-3 and α-SMA gene expression. CONCLUSIONS: The administration of antihistamines resulted in changes in the rat salivary glands, which could be due to the induction of oxidative stress and the resultant apoptotic effect. These changes were suggested to occur mainly through action on muscarinic receptors; yet, action on histamine receptors could not be excluded. However; these effects were less marked with the second-generation antihistamine.
Subject(s)
Actins , Caspase 3 , Nitric Oxide Synthase Type II , Parotid Gland , Animals , Rats , Male , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide Synthase Type II/genetics , Parotid Gland/drug effects , Parotid Gland/metabolism , Caspase 3/metabolism , Actins/metabolism , Actins/genetics , Cetirizine/pharmacology , Histamine H1 Antagonists/pharmacologyABSTRACT
Testicular torsion is an urological emergency and can lead to ischemia damage and testicular loss if not diagnosed in time. Proanthocyanidin is reported to have anti-inflammatory and antioxidant properties. The current study aimed to examine the possible effects of proanthocyanidin (P) on the testis in torsion/detorsion (T/D)-induced testicular ischemia/reperfusion (I/R) injury in rats. Forty rats were divided into four groups (n=10 for each): sham-operated (sham), I/R, I/R + P100 (100â¯mg/kg, 30â¯min before torsion), and I/R + P200 (200â¯mg/kg, 30â¯min before torsion). Testicular T/D was performed on the left testicle by 3â¯hours of torsion at 720° clockwise, followed by 3â¯hours of detorsion. In the I/R group, an increase in malondialdehyde (MDA) levels and a decrease in glutathione (GSH), vitamin C (Vit C), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PD) values were determined compared to the sham group (p<0.001). Moreover, an increase in the expression of cleaved caspase-3 and Bcl2-associated X protein (Bax), a decrease in the expression of B-cell lymphoma 2 (Bcl-2) and proliferating cell nuclear antigen (PCNA) were detected in the I/R group (p<0.001). Histopathologically, it was determined that the Johnsen and Cosentino scores of the testicles were irregular in the I/R group (p<0.001). Proanthocyanidin treatment caused a decrease in MDA, cleaved caspase-3 and Bax levels and an increase in GSH, Vit C, GPx, G6PD, Bcl-2 and PCNA values. Additionally, Johnsen and Cosentino rearranged the scores. The present findings revealed the protective and curative effects of proanthocyanidin in organ damage due to testicular torsion/detorsion-induced ischemia/reperfusion with their antioxidative and antiapoptotic properties.
Subject(s)
Proanthocyanidins , Reperfusion Injury , Spermatic Cord Torsion , Testis , Animals , Male , Proanthocyanidins/pharmacology , Reperfusion Injury/drug therapy , Reperfusion Injury/pathology , Reperfusion Injury/metabolism , Spermatic Cord Torsion/metabolism , Spermatic Cord Torsion/complications , Spermatic Cord Torsion/drug therapy , Spermatic Cord Torsion/pathology , Rats , Testis/metabolism , Testis/drug effects , Testis/pathology , Apoptosis/drug effects , Antioxidants/pharmacology , Oxidative Stress/drug effects , Glutathione/metabolism , Malondialdehyde/metabolism , Glutathione Peroxidase/metabolism , bcl-2-Associated X Protein/metabolism , Caspase 3/metabolism , Rats, WistarABSTRACT
Acute lung injury is one of the most serious complications of sepsis, which is a common critical illness in clinic. This study aims to investigate the role of caspase-3/ gasdermin-E (GSDME)-mediated pyroptosis in sepsis-induced lung injury in mice model. Cecal ligation (CLP) operation was used to establish mice sepsis-induced lung injury model. Lung coefficient, hematoxylin and eosin staining and transmission electron microscopy were used to observe the lung injury degree. In addition, caspase-3-specific inhibitor Z-DEVD-FMK and GSDME-derived inhibitor AC-DMLD-CMK were used in CLP model, caspase-3 activity, GSDME immunofluorescence, serum lactate dehydrogenase (LDH) and interleukin-6 (IL-6) levels, TUNEL staining, and the expression levels of GSDME related proteins were detected. The mice in CLP group showed the increased expressions of cleaved-caspase-3 and GSDME-N terminal, destruction of lung structure, and the increases of LDH, IL-6, IL-18 and IL-1ß levels, which were improved in mice treated with Z-DEVD-FMK or AC-DMLD-CMK. In conclusion, caspase-3/GSDME mediated pyroptosis is involved in the occurrence of sepsis-induced lung injury in mice model, inhibiting caspase-3 or GSDME can both alleviate lung injury.
Subject(s)
Acute Lung Injury , Caspase 3 , Disease Models, Animal , Pyroptosis , Sepsis , Animals , Pyroptosis/drug effects , Sepsis/complications , Mice , Caspase 3/metabolism , Acute Lung Injury/pathology , Male , Mice, Inbred C57BL , Interleukin-6/metabolism , Caspase Inhibitors/pharmacology , Lung/pathology , Lung/metabolism , Oligopeptides/pharmacology , GasderminsABSTRACT
Corneal endothelial cells (CE) are critical for the cornea's transparency. For severe corneal damage, corneal tissue transplantation is the most promising option for restoring vision. However, CE apoptotic cell death occurs during the storage of donor corneas for transplantation. This study used small interfering (si)RNA-mediated silencing of pro-apoptotic proteins as a novel strategy to protect CE against apoptosis. Therefore, the pro-apoptotic proteins Bax and Bak were silenced in the human corneal endothelial cell line (HCEC-12) by transfection with Accell™siRNA without any adverse effects on cell viability. When apoptosis was induced, e.g., etoposide, the caspase-3 activity and Annexin V-FITC/PI assay indicated a significantly reduced apoptosis rate in Bax+Bak-siRNA transfected HCECs compared to control (w/o siRNA). TUNEL assay in HCECs exposed also significantly lower cell death in Bax+Bak-siRNA (7.5%) compared to control (w/o siRNA: 32.8%). In ex vivo donor corneas, a significant reduction of TUNEL-positive CEs in Bax+Bak-siRNA corneas (8.1%) was detectable compared to control-treated corneas (w/o siRNA: 27.9%). In this study, we demonstrated that suppressing pro-apoptotic siRNA leads to inhibiting CE apoptosis. Gene therapy with siRNA may open a new translational approach for corneal tissue treatment in the eye bank before transplantation, leading to graft protection and prolonged graft survival.
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The development of selective and sensitive probes for monitoring caspase-3 activity-a critical enzyme involved in apoptosis-remains an area of significant interest in biomedical research. Herein, we report the synthesis and characterisation of a novel ratiometric fluorescent probe, Ac-DEVD-PABC-Naph, designed to detect caspase-3 activity. The probe utilises a 1,8-naphthalimide fluorophore covalently linked to a peptide sequence via a self-immolative p-aminobenzyl alcohol (PABA) linker. Upon enzymatic cleavage by caspase-3, the probe undergoes spontaneous degradation, releasing the free naphthalimide fluorophore, resulting in a ratiometric change in fluorescence emission. Spectroscopic studies revealed a time-dependent ratiometric fluorescent response, demonstrating the probe's ability to visualise caspase-3 activity with high sensitivity. Enzyme kinetics such as K m (Michaelis constant), k cat (turnover number), and LOD (Limit of Detection) were obtained, suggesting that the probe possesses comparable kinetic data to other probes in literature, but with the added benefits of ratiometric detection. Selectivity studies also demonstrated the probe's specificity for caspase-3 over other endogenous species and enzymes. Ac-DEVD-PABC-Naph may be a promising tool for the quantitative detection and fluorescent visualisation of caspase-3 activity in biological systems, with potential applications in apoptosis research and drug development.
ABSTRACT
BACKGROUND: Hepatotoxicity associated with methotrexate (MTX) is mainly due to disruption of redox balance and development of oxidative injury to hepatocytes. Melatonin (MLT) is a potent antioxidant and regulates wide range of biological functions, processes and utilized as adjuvant for number of medical applications. The current study investigated the mitigating effect of MLT on the MTX-induced hepatotoxicity. METHODS AND RESULTS: Adult male rats received MLT (25 mg/kg, orally) for seven days flowed by single injection of MTX (20 mg/kg, ip) then treat with MLT continued for additional 7 days. The present result showed MLT treatment mitigated histopathological changes in the liver that associated with normalization of ALT and AST activity as well as bilirubin, albumin and alfa-fetoprotein levels in serum of MLT + MTX-treated rat to comparable control level. MLT treatment significantly reduced MDA content and myeloperoxidase activity while enhanced the activity of superoxide dismutase, catalase and glutathione content in the liver indicating the empowerment of the antioxidant status. Amelioration of MLT-induced oxidative stress resulted in a reduction in the inflammatory response due to antioxidant restoration and inhibited apoptosis indicated by downregulation of caspase-3 expression. The replenishment of antioxidant content powers the defense system of the hepatocytes. As a result, apoptosis is reduced which might be due to the ability of MLT protect DNA integrity thus maintaining hepatocyte functions and structure. Consequently, liver histology was protected. CONCLUSIONS: In summary, MLT modulates liver function and structure by orchestrating linked processes, including redox balance, inflammatory response, suppression of caspase-3, and DNA damage.
Subject(s)
Antioxidants , Apoptosis , Chemical and Drug Induced Liver Injury , Hepatocytes , Liver , Melatonin , Methotrexate , Oxidative Stress , Animals , Methotrexate/adverse effects , Methotrexate/toxicity , Melatonin/pharmacology , Rats , Male , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/drug therapy , Oxidative Stress/drug effects , Liver/drug effects , Liver/metabolism , Liver/pathology , Antioxidants/pharmacology , Antioxidants/metabolism , Apoptosis/drug effects , Hepatocytes/drug effects , Hepatocytes/metabolism , Superoxide Dismutase/metabolism , Glutathione/metabolism , Catalase/metabolismABSTRACT
PURPOSE: This study aimed to investigate the protective or therapeutic effect of thymoquinone (TQ) in a retinal degeneration rat model and its relationships with the retina ultrastructure, heme oxygenase 1 (HO-1), caspase-3, and RPE65 expressions and to determine whether TQ has a therapeutic effect at the biochemical level. METHODS: A total of 25 adult Wistar albino rats were divided into the following treatment groups: saline (control: CONT), CO (corn oil), sodium iodate (SI), TQ + SI, and SI + TQ injection groups. Retina morphology, RPE65, HO-1, and caspase-3 expression levels were evaluated using immunohistochemistry, and optical density was determined using ImageJ. Ultrastructural evaluations were performed with electron microscopy. Thiol-disulfide homeostatic parameters were examined in serum samples. RESULTS: Outer nuclear layer (ONL) thickness was significantly higher in the SI + TQ group compared to the SI group. The RPE65 expression significantly decreased in the SI group compared with the CONT and CO groups. A significant increase in RPE65 expression level and a significant decrease in caspase-3 expression level were found in the SI + TQ group compared with the SI group. The increase in HO-1 expression level was significantly higher in the TQ treatment groups, particularly in the SI + TQ group. In the SI and TQ + SI groups, the ONL thickness significantly decreased with a significant increase in caspase-3 expression compared to the CONT and CO groups. In the treatment groups, decreased organelle damage was observed on electron microscopy. In the SI + TQ group, the disulfide/native thiol and disulfide/total thiol ratios were significantly lower than all other groups, while the native/total thiol ratio was significantly higher than the other experimental groups. CONCLUSIONS: The present study provides evidence that continuous TQ treatment can increase HO-1 and RPE65 expression and decrease apoptosis (caspase-3 levels), thereby preserving the retina at the ultrastructural level. Moreover, TQ administration can maintain thiol/disulfide homeostasis in SI-induced retinal degeneration-modelled rats.
ABSTRACT
The amyloid-beta peptide (Aß) is the neurotoxic component in senile plaques of Alzheimer's disease (AD) brains. Previously we have reported that Aß toxicity is mediated by the induction of sonic hedgehog (SHH) to trigger cell cycle re-entry (CCR) and apoptosis in post-mitotic neurons. Basella alba is a vegetable whose polysaccharides carry immunomodulatory and anti-cancer actions, but their protective effects against neurodegeneration have never been reported. Herein, we tested whether polysaccharides derived from Basella alba (PPV-6) may inhibit Aß toxicity and explored its underlying mechanisms. In differentiated rat cortical neurons, Aß25-35 reduced cell viability, damaged neuronal structure, and compromised mitochondrial bioenergetic functions, all of which were recovered by PPV-6. Immunocytochemistry and western blotting revealed that Aß25-35-mediated induction of cell cycle markers including cyclin D1, proliferating cell nuclear antigen (PCNA), and histone H3 phosphorylated at Ser-10 (p-Histone H3) in differentiated neurons was all suppressed by PPV-6, along with mitigation of caspase-3 cleavage. Further studies revealed that PPV-6 inhibited Aß25-35 induction of SHH; indeed, PPV-6 was capable of suppressing neuronal CCR and apoptosis triggered by the exogenous N-terminal fragment of sonic hedgehog (SHH-N). Our findings demonstrated that, in the fully differentiated neurons, PPV-6 exerts protective actions against Aß neurotoxicity via the downregulation of SHH to suppress neuronal CCR and apoptosis.
Subject(s)
Amyloid beta-Peptides , Apoptosis , Cell Cycle , Hedgehog Proteins , Neurons , Polysaccharides , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/toxicity , Hedgehog Proteins/metabolism , Animals , Neurons/drug effects , Neurons/metabolism , Apoptosis/drug effects , Rats , Polysaccharides/pharmacology , Polysaccharides/chemistry , Cell Cycle/drug effects , Peptide Fragments , Cell Survival/drug effects , Neuroprotective Agents/pharmacologyABSTRACT
This study explores the application of the RIP3-caspase3-assay in heterogeneous spheroid cultures to analyze cell death pathways, emphasizing the nuanced roles of apoptosis and necroptosis. By employing directly conjugated monoclonal antibodies, we provide detailed insights into the complex mechanisms of cell death. Our findings demonstrate the assay's capability to differentiate between RIP1-independent apoptosis, necroptosis, and RIP1-dependent apoptosis, marking a significant advancement in organoid research. Additionally, we investigate the effects of TNFα on isolated intestinal epithelial cells, revealing a concentration-dependent response and an adaptive or threshold reaction to TNFα-induced stress. The results indicate a preference for RIP1-independent cell death pathways upon TNFα stimulation, with a notable increase in apoptosis and a secondary role of necroptosis. Our research underscores the importance of the RIP3-caspase3-assay in understanding cell death mechanisms in organoid cultures, offering valuable insights for disease modeling and the development of targeted therapies. The assay's adaptability and robustness in spheroid cultures enhances its potential as a tool in personalized medicine and translational research.