Caspase-3 Research Articles

Açaí berry ameliorates cognitive impairment by inhibiting NLRP3/ASC/CASP axis in STZ-induced diabetic neuropathy in mice.

Diabetes complications such as diabetic peripheral neuropathy (DPN) are linked to morbidity and mortality. Peripheral nerve damages in DPN are accompanied by discomfort, weakness, and sensory loss. Some drugs may demonstrate their therapeutic promise by reducing neuroinflammation, but they have side effects. Based on these considerations, the objective of this study was to examine the beneficial properties of açaí berry in a mouse model of DPN generated by injection of streptozotocin (STZ). Açaí berry was given orally to diabetic and control mice every day beginning 2 wk after STZ injection. The animals were euthanized after 16 wk, and tissues from the spinal cord and sciatic nerve and urine were taken. Our findings showed that daily treatment of açaí berry at a dose of 500 mg/kg was able to prevent behavioral changes as well as mast cell activation and nerve deterioration via NOD-like receptor family pyrin-domain-containing-3 (NLRP3)/apoptosis-associated speck-like protein containing a card (ASC)/caspase (CASP) regulation after diabetes induction.NEW & NOTEWORTHY Our research shows that açaí berry reduces mast cells degranulation and histological damage in diabetic neuropathy, improves physiological defense against reactive oxygen species, modulates the NLRP3/ASC/CASP axis, and ameliorates inflammation and oxidative stress. Diet could help treatment for diabetic peripheral neuropathy.

Effects of astaxanthin on expression of apoptosis and oxidative stress related genes in H2O2 induced oxidative stress BE(2)-C human neuroblastoma cell line

Antioxidants may affect the apoptosis induced by oxidative stress experimental models. The present study was conducted to investigate the effects of astaxanthin on expression of apoptosis and oxidative stress-related genes in H2O2 induced oxidative stress BE(2)-C human neuroblastoma cell line. This experimental study consisted of six groups including control, H2O2 induced oxidative stress control, 100 mM vitamin C intervention, 25 μM astaxanthin intervention (Ax1), 50 μM astaxanthin intervention (Ax2) and 100 μM astaxanthin intervention (Ax3). Real-time PCR was used to study the expression of BAX, BCL2, Caspase3 (CAS3), P53, peroxisome proliferator-activated receptor γ (PPARγ), superoxide dismutase (SOD), glutathione peroxidase 1 (GPX), catalase (CAT) and nuclear factor erythroid 2–related factor 2 (NRF2). According to the results, among the apoptosis-related genes, CAS3 was down-regulated in groups vitamin C, Ax1 and Ax2 compared with H2O2 group, while P53 was down-regulated only in group vitamin C (P < 0.05). Among the oxidative stress-related genes, GPX was up-regulated in groups Ax1, Ax2 and Ax3 compared with H2O2 group, while all the experimental groups showed up-regulation for CAT and NRF2 (P < 0.05). In conclusion, astaxanthin as a powerful antioxidant could inhibit apoptosis via amelioration of CAS3 gene which might be through amelioration of some antioxidant-related genes.

Cholinergic-like neurons and cerebral spheroids bearing the PSEN1 p.Ile416Thr variant mirror Alzheimer's disease neuropathology

Familial Alzheimer’s disease (FAD) is a complex neurodegenerative disorder for which there are no therapeutics to date. Several mutations in presenilin 1 (PSEN 1), which is the catalytic component of γ-secretase complex, are causal of FAD. Recently, the p.Ile416Thr (I416T) PSEN 1 mutation has been reported in large kindred in Colombia. However, cell and molecular information from I416T mutation is scarce. Here, we demonstrate that menstrual stromal cells (MenSCs)-derived planar (2D) PSEN 1 I416T cholinergic-like cells (ChLNS) and (3D) cerebral spheroids (CSs) reproduce the typical neuropathological markers of FAD in 4 post-transdifferentiating or 11 days of transdifferentiating, respectively. The models produce intracellular aggregation of APPβ fragments (at day 4 and 11) and phosphorylated protein TAU at residue Ser202/Thr205 (at day 11) suggesting that iAPPβ fragments precede p-TAU. Mutant ChLNs and CSs displayed DJ-1 Cys106-SO3 (sulfonic acid), failure of mitochondria membrane potential (ΔΨm), and activation of transcription factor c-JUN and p53, expression of pro-apoptotic protein PUMA, and activation of executer protein caspase 3 (CASP3), all markers of cell death by apoptosis. Moreover, we found that both mutant ChLNs and CSs produced high amounts of extracellular eAβ42. The I416T ChLNs and CSs were irresponsive to acetylcholine induced Ca2+ influx compared to WT. The I416T PSEN 1 mutation might work as dominant-negative PSEN1 mutation. These findings might help to understanding the recurring failures of clinical trials of anti-eAβ42, and support the view that FAD is triggered by the accumulation of other intracellular AβPP metabolites, rather than eAβ42.

VPS41-mediated incomplete autophagy aggravates cadmium-induced apoptosis in mouse hepatocytes

Exposure to cadmium (Cd), an environmental heavy metal contaminant, is a serious threat to global health that increases the burden of liver diseases. Autophagy and apoptosis are important in Cd-induced liver injury. However, the regulatory mechanisms involved in the progression of Cd-induced liver damage are poorly understood. Herein, we investigated the role of vacuolar protein sorting 41 (VPS41) in Cd-induced autophagy and apoptosis in hepatocytes. We used targeted VPS41 regulation to elucidate the mechanism of Cd-induced hepatotoxicity. Our data showed that Cd triggered incomplete autophagy by downregulating VPS41, aggravating Cd-induced hepatocyte apoptosis. Mechanistically, Cd-induced VPS41 downregulation interfered with the mTORC1-TFEB/TFE3 axis, leading to an imbalance in autophagy initiation and termination and abnormal activation of autophagy. Moreover, Cd-induced downregulation of VPS41 inhibited autophagosome–lysosome fusion, leading to blocked autophagic flux. This triggers incomplete autophagy, which causes excessive P62 accumulation, accelerating Caspase-9 (CASP9) cleavage. Incomplete autophagy blocks clearance of cleaved CASP9 (CL-CASP9) via the autophagic pathway, promoting apoptosis. Notably, VPS41 overexpression alleviated Cd-induced incomplete autophagy and apoptosis, independent of the homotypic fusion and protein sorting complex. This study provides a new mechanistic understanding of the relationship between autophagy and apoptosis, suggesting that VPS41 is a new therapeutic target for Cd-induced liver damage.

Ononin Relieves the Thyroid Cancer Progression through Targeting the Caspase 3 and CD274 Expression Levels.

Thyroid cancer (TC) is the most common malignant tumor of endocrine system and head and neck. Ononin is an isoflavone component, which exhibited great antioxidant and anti-inflammatory activities. This study was conducted to explore the functions of ononin in the TC progression. The cell counting kit-8 (CCK8) assay was applied for the cell viability determination. The cell death and apoptosis rate were analyzed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining and flow cytometry. The quantitative real-time PCR (qRT-PCR) and Western blot assays were performed for the relative expressions determination. Lactate dehydrogenase (LDH) release assay was used to assess cytotoxicity. Ononin treatment prominently inhibited the cell viability and induced the cell apoptosis of the TC cells. Besides, caspase 3 (CASP3) was down-regulated and CD274 was up-regulated in TC. Ononin treatment prominently decreased the CD274 levels and increased the CASP3 levels in the TC cells. Additionally, ononin treatment dramatically enhanced the LDH release of the cytotoxicity of T cells. What is more, CASP3 overexpression or CD274 knockdown promoted the role of ononin in TC cells. Ononin treatment induced the cell death of the TC cells through regulating the CASP3 and CD274 expressions.

BIIB021, an orally available and small-molecule inhibitor of HSP90, activates intrinsic apoptotic pathway in human cervical adenocarcinoma cell line (HeLa).

Heat shock protein 90 (HSP90) is a highly conserved ATP-dependent chaperone protein that plays a vital role in tumorigenesis. This study aims to investigate the apoptosis inducer role of BIIB021 (orally available HSP90 inhibitors) compound via inhibition of HSP90 activity in the human cervical cancer cell line (HeLa). The anticancer potential of BIIB021 was determined by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)] cell proliferation assay against the human cervical cancer cell line (HeLa). ATPase and luciferase aggregation assays were carried out to detect the HSP90 inhibitor potential of BIIB021. To determine the antiproliferative mechanism of the BIIB021, the expression level of the pro-apoptotic and antiapoptotic markers was determined by reverse transcription polymerase chain reaction (RT-PCR) and ELISA experiments. BIIB021 exhibited a cytotoxic effect on HeLa cell proliferation and the inhibitory concentration (IC)50 dose of BIIB021 was found to be 14.79 nM at 48 h. BIIB021 decreased the ATP hydrolysis rate of HSP90 and blocked the refolding of the desaturated luciferase in the presence of ATP. To understand the antiproliferative mechanism of the BIIB021 in HeLa cells, the mRNA and protein expression levels of the apoptotic markers [BCL-2 associated X (BAX), B-cell lymphoma 2 (BCL-2), cytochrome-c (CYT-c), and caspase-3 (CAS-3)] were determined by RT-PCR and ELISA experiments. The results obtained indicated that BIIB021 decreased BCL-2 levels and increased BAX, CYT-c, and CAS-3 levels in human cervical cancer cells. These results confirmed that BIIB021 inhibited the chaperone activity of HSP90, resulting in anti-proliferating effects in cervical cancer cells via the induction of the intrinsic apoptotic pathways.

Gallic acid and metformin co-administration reduce oxidative stress, apoptosis and inflammation via Fas/caspase-3 and NF-κB signaling pathways in thioacetamide-induced acute hepatic encephalopathy in rats

BackgroundHepatic encephalopathy (HE) is a consequence of chronic or acute liver diseases. This study evaluates the combined effect of gallic acid (GA), and metformin (Met) on the liver and brain damage associated with HE.MethodsAcute HE was induced by a single dose of thioacetamide (TAA) (300 mg/kg) as an I.P. injection. Treated groups received GA group (100 mg/kg/day, p.o), Met (200 mg/kg/day, p.o), or their combination for 25 consecutive days before TAA injection.ResultsThe administration of TAA induced various biochemical and histopathological alterations. In contrast, treatment with GA either alone or combined with Met resulted in improved liver functions by the significant reduction in serum ALT, AST, and ALP activities, and ammonia levels. Inflammatory mediators; TNF-α, IL-6, and NFkβ levels were decreased by these treatments as well as apoptotic cascade via down-regulation of FAS and caspase-3 (CASP-3) expression in hepatic tissues. Furthermore, GA and Met either alone or combined protected the liver and brain tissues from damage by increased glutathione concentration while decreasing malondialdehyde. In addition, it was accompanied by the improvement of the brain neurotransmitter profile via the restoration of norepinephrine, dopamine, and serotonin levels. Based on our data, this is the first study to report a novel combined hepatoprotective and cognitive enhancing effect of GA and Met against TAA-induced acute liver and brain injury.ConclusionGA and Met combination resulted in a prominent improvement in HE complications, relative to monotherapy. Both agents potentiated the antioxidant, anti-inflammatory, and anti-apoptotic effects of each other.

Caspase activation in tumour-infiltrating lymphocytes is associated with lymph node metastasis in oral squamous cell carcinoma.

Oral squamous cell carcinomas (OSCCs) are genetically heterogeneous and exhibit diverse stromal and immune microenvironments. Acquired resistance to standard chemo-, radio-, and targeted therapies remains a major hurdle in planning effective treatment modalities for OSCC patients. Since Caspase 8 (CASP8) is frequently mutated in OSCCs, we were interested to explore a potential interaction between tumour-infiltrating lymphocytes (TILs) and CASP8 activation using high-content image analysis of human tumour (n = 32) sections. Despite the lymphocyte-rich tumour microenvironment, we observed lower activation of CASP8 (0-10% of tumour area) and its downstream effector CASP3 (0-6%) in tumours than in normal oral epithelium. Conversely, we found apoptosis was high for all the lymphocyte subtypes examined (38-52% of lymphocytes within tumour islands). Tumours with higher Fas ligand (FasL) expression had a significantly higher proportion of cleaved CASP3/8 positive cytotoxic T cells within the tumour islands (p = 0.05), and this was associated with the presence of lymph node metastatic disease [odds ratio: 1.046, 95% confidence interval (1.002-1.091), p = 0.039]. Our finding of extensive activation of the extrinsic pathway of apoptosis in TILs, together with evidence of higher FasL in CASP8 mutated tumours, may be useful in predicting the course of disease in individual patients. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The autophagic protein FYCO1 controls TNFRSF10/TRAIL receptor induced apoptosis and is inactivated by CASP8 (caspase 8)

ABSTRACT Apoptosis is a tightly controlled cell death program executed by proteases, the so-called caspases. It plays an important role in tissue homeostasis and is often dysregulated in cancer. Here, we identified FYCO1, a protein that promotes microtubule plus end-directed transport of autophagic and endosomal vesicles as a molecular interaction partner of activated CASP8 (caspase 8). The absence of FYCO1 sensitized cells to basal and TNFSF10/TRAIL-induced apoptosis by receptor accumulation and stabilization of the Death Inducing Signaling Complex (DISC). Loss of FYCO1 resulted in impaired transport of TNFRSF10B/TRAIL-R2/DR5 (TNF receptor superfamily member 10b) to the lysosomes in TNFSF10/TRAIL-stimulated cells. More in detail, we show that FYCO1 interacted via its C-terminal GOLD domain with the CCZ1-MON1A complex, which is necessary for RAB7A activation and for the fusion of autophagosomal/endosomal vesicles with lysosomes. We demonstrated that FYCO1 is a novel and specific CASP8 substrate. The cleavage at aspartate 1306 resulted in the release of the C-terminal GOLD domain, inactivating FYCO1 function, and allowing for the progression of apoptosis. Furthermore, the lack of FYCO1 resulted in a stronger and prolonged formation of the TNFRSF1A/TNF-R1 signaling complex. Thus, FYCO1 limits the ligand-induced and steady-state signaling of TNFR-superfamily members, providing a control mechanism that fine-tunes both apoptotic and inflammatory answers. Abbreviations: AP: affinity purification; CHX: cycloheximide; co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; DISC: death-inducing signaling complex; DR: death receptors; doxy: doxycycline; GEF: guanine nucleotide exchange factor; ind: inducible; KD: knockdown; KO: knockout; MS: mass spectrometry; shRNA: short hairpin RNA; siRNA: small interfering RNA; TIP: two-step co-immunoprecipitation; WB: western blot.

Anti-inflammatory material basis and mechanism of Artemisia stolonifera based on UPLC-Q-TOF-MS combined with network pharmacology and molecular docking

This study aimed to explore the anti-inflammatory material basis and molecular mechanism of Artemisia stolonifera based on the analysis of the chemical components in different extracted fractions of A. stolonifera and their antioxidant and anti-inflammatory effects in combination with network pharmacology and molecular docking. Thirty-two chemical components were identified from A. stolonifera by ultra-performance liquid chromatography coupled to tandem quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS). Among them, there were 7, 21 and 22 compounds in water, n-butanol and ethyl acetate fractions, respectively. The antio-xidant capacity of different extracted fractions was evaluated by measuring their scavenging ability against 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl(DPPH) and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid)(ABTS) free radicals and total antioxidant capacity [ferric reducing antioxidant power(FRAP) assay]. The inflammatory model of RAW264.7 cells was induced by lipopolysaccharide(LPS), and the levels of nitrite oxide(NO), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) in the supernatant and the mRNA expression of related inflammatory factors in cells were used to evaluate the anti-inflammatory effects. The results revealed that ethyl acetate fraction of A. stolonifera was the optimal antioxidant and anti-inflammatory fraction. By network pharmacology, it was found that flavonoids such as rhamnazin, eupatilin, jaceosidin, luteolin and nepetin could act on key targets such as TNF, serine/threonine protein kinase 1(AKT1), tumor protein p53(TP53), caspase-3(CASP3) and epidermal growth factor receptor(EGFR), and regulate the phosphatidylinositol-3-kinase-protein kinase B(PI3K-AKT) and mitogen-activated protein kinase(MAPK) signaling pathways to exert the anti-inflammatory effects. Molecular docking further indicated excellent binding properties between the above core components and core targets. This study preliminarily clarified the anti-inflammatory material basis and mechanism of ethyl acetate fraction of A. stolonifera, providing a basis for the follow-up clinical application of A. stolonifera and drug development.

Exploring the Antidiabetic Effect of Lupenone in Rats with Type 1 Diabetes and its Underlying Mechanism Based on Network Pharmacology

Lupenone has been reported to possess numerous medicinal values and gives a positive antidiabetic effect. But the mechanism of preventing and treating type 1 diabetes has not been elucidated in type 1 diabetic rats. This study investigated the effects and mechanism of action of lupenone in preventing and treating type 1 diabetes by network pharmacology and diabetic rats. The blood glucose, glycosylated hemoglobin (HbA1c), insulin, and inflammatory factors in the pancreas of rats with type 1 diabetes were measured, and histopathological changes were observed after treatment with lupenone. The pharmacological network of ‘component-target-disease’ was constructed on diabetic rats. Gene function enrichment, the Kyoto Encyclopedia of Genes and Genomes pathway analysis, and molecular docking were performed. The results showed that lupenone can decrease fasting blood glucose and HbA1c levels, increase insulin content and interleukin (IL)-4, IL-10, and decrease IL-6, transforming growth factor β and tumor necrosis factor α levels in the pancreas. Furthermore, ten targets were identified, and 50 signal pathways closely related to type 1 diabetes and inflammation were screened by network pharmacology, including insulin resistance, type II diabetes, type I diabetes, insulin signal pathway, mitogen activated protein kinase (MAPK) signal pathway, and tumor necrosis factor (TNF) signal pathway. The docking affinity of potential targets and lupenone were between -3.3 and -9.8, among which caspase-3 (CASP3), cyclin-dependent kinase 4 (CDK4), inhibitor of kappaB kinase beta (IKBKB), transforming growth factor beta-1 (TGFB1), and TNF had high binding abilities. Thus, lupenone has the potential to be developed as a new drug for treating type 1 diabetes.

Novel promising benzoxazole/benzothiazole-derived immunomodulatory agents: Design, synthesis, anticancer evaluation, and in silico ADMET analysis.

Eleven novel benzoxazole/benzothiazole-based thalidomide analogs were designed and synthesized to obtain new effective antitumor immunomodulatory agents. The synthesized compounds were evaluated for their cytotoxic activities against HepG-2, HCT-116, PC3, and MCF-7 cells. Generally, the open analogs with semicarbazide and thiosemicarbazide moieties (10, 13a-c, 14, and 17a,b) exhibited higher cytotoxic activities than derivatives with closed glutarimide moiety (8a-d). In particular, compound 13a (IC50 = 6.14, 5.79, 10.26, and 4.71 µM against HepG-2, HCT-116, PC3, and MCF-7, respectively) and 14 (IC50 = 7.93, 8.23, 12.37, and 5.43 µM, respectively) exhibited the highest anticancer activities against the four tested cell lines. The most active compounds 13a and 14 were further evaluated for their in vitro immunomodulatory activities on tumor necrosis factor-alpha (TNF-α), caspase-8 (CASP8), vascular endothelial growth factor (VEGF), and nuclear factor kappa-B p65 (NF-κB p65) in HCT-116 cells. Compounds 13a and 14 showed a remarkable and significant reduction in TNF-α. Furthermore, they showed significant elevation in CASP8 levels. Also, they significantly inhibited VEGF. In addition, compound 13a showed significant decreases in the level of NF-κB p65 while compound 14 demonstrated an insignificant decrease with respect to thalidomide. Moreover, our derivatives exhibited good in silico absorption, distribution, metabolism, elimination, toxicity (ADMET) profiles.

Study on the mechanism of Lily in the treatment of viral pneumonia based on network pharmacology analysis

Background: Viral pneumonia poses an increasing threat to human life and health, and the treatment of viral pneumonia with single traditional Chinese medicine is a frontier trend in clinical research at present. Objective: Based on network pharmacology, this study aimed at exploring the potential targets and mechanisms of Lilium brownii in treating viral pneumonia. Methods: Traditional Chinese Medicine Systems Pharmacology (TCMSP) (https://tcmspw.com/index.php), the database and analysis platform of traditional Chinese medicine, was employed to retrieve and convert the active components and component targets of Lily. Then, the targets identified in the TCMSP database, were translated into the specific list of UniProt database (https://www.uniprot.org/). Meanwhile, GeneCards database was utilized to obtain the targets associated with viral pneumonia, (https://www.genecards.org). Subsequently, we employed the Venny2.1 database (https://bioinfogp.cnb.csic.es/tools/venny/) for the construction of network and the analysis of active ingredients as well as key targets. Results: Six active components, 447 action targets, 3,473 disease targets and 4 drug cross targets were obtained. The top 10 GO biological processes, 10 GO cell components, 10 GO molecular functions and the top 10 KEGG signal pathways, as well as key molecules were identified. Of these, Lily may act on three key targets, including the apoptosis regulator BCL2, Caspase-3 (CASP3), Caspase-8 (CASP8), and p53 signal pathway. Conclusions: Lily may play a regulatory role in the manifestation of viral pneumonia, as it modulates apoptosis processes across diverse species and influences the p53 signaling pathway. These discoveries establish a foundation for prospective experimental validation.

Review of molecular biological studies on acute lymphoblastic leukemia treated by modified shengmaiyin.

The objective was to explore the pharmacological mechanism of modified shengmaiyin (MSMY) in the treatment of acute lymphoblastic leukemia (ALL) by network pharmacology analysis. The effective components and predicted targets of MSMY were collected from TCMSP and Swiss target prediction databases, and the related targets of ALL were screened by GeneCards and DisGeNET. The core targets and related signaling pathways of MSMY active ingredients for the treatment of ALL were predicted by protein-protein interaction network (PPI), gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis. We identified 172 potential targets for the active components of MSMY, 538 disease targets associated with ALL, and 59 common gene targets. PPI network showed that 27 targets such as triptolide, RAC-alpha serine/threonine-protein kinase (AKT1), vascular endothelial growth factor A and Caspase-3 (CASP3) were the core targets. KEGG enrichment analysis related signaling pathways included cancer pathway, phosphatidylinositol 3 kinase, PI-3K/protein kinase B (PI3K-Akt) signaling pathway, apoptosis and mitogen-activated protein kinase (MAPK) signaling pathway and IL-17 signaling pathway. The effective active components and potential therapeutic targets of MSMY in the treatment of ALL were initially identified by comprehensive network pharmacology, which provides a theoretical basis for further study of the material basis and molecular mechanism of MSMY in the treatment of ALL.

Apoptosis-targeted gene therapy for non-small cell lung cancer using chitosan-poly-lactic-co-glycolic acid -based nano-delivery system and CASP8 and miRs 29A-B1 and 34A.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide, with resistance to apoptosis being a major driver of therapeutic resistance and aggressive phenotype. This study aimed to develop a novel gene therapy approach for NSCLC by targeting resistance to apoptosis. Loss of function mutations of caspase 8 (CASP8) and downregulation of microRNAs (miRs) 29A-B1 and 34A were identified as key contributors to resistance to apoptosis in NSCLC. A biodegradable polymeric nano-gene delivery system composed of chitosan-poly-lactic-co-glycolic acid was formulated to deliver initiator CASP8 and miRs 29A-B1 and 34A. The nano-formulation efficiently encapsulated the therapeutic genes effectively internalized into NSCLC cells and induced significant apoptosis. Evaluation of the nano-formulation in A549 tumor spheroids showed a significant increase in apoptosis within the core of the spheroids, suggesting effective penetration into the spheroid structures. We provide a novel nano-formulation that demonstrate therapeutic potential for suicidal gene therapy in NSCLC.

Potential Mechanisms of Metformin-Induced Apoptosis in HeLa Cells

Metformin is a traditional antidiabetic drug that also shows potential antitumor effects in cervical cancer. However, some of its apoptosis-related mechanisms are still unclear. In this study, flow cytometry, western blotting, and RNA sequencing (RNA-seq) were used to evaluate the molecular mechanisms of metformin in HeLa cells. The results showed that metformin inhibited cell viability and promoted apoptosis, the protein expression level of Caspase-3 (CASP3) was increased and that of BCL-2 was decreased in HeLa cells treated with metformin. The RNA-seq results indicated a total of 239 differentially expressed genes between the metformin and control check (CK) groups, with 136 genes upregulated and 103 genes downregulated, and 14 of them were found to be associated with apoptosis signaling pathways. The DDIT3 and HRK genes were robustly upregulated in HeLa cells by the endoplasmic reticulum (ER) stress and the mitochondrial pathway of apoptosis. Metformin also affects the expression of PPP2R5C, PPP2R5A, and RRAGA, which participate in biological processes such as PI3K-AKT, mTOR, and AMPK signaling pathways. Metformin mediates the expression of related genes to induce apoptosis.

Cannabidiol Protects Dopaminergic-like Neurons against Paraquat- and Maneb-Induced Cell Death through Safeguarding DJ-1CYS106 and Caspase 3 Independently of Cannabinoid Receptors: Relevance in Parkinson's Disease.

Parkinson's disease (PD), a progressive neurodegenerative movement disorder, has reached pandemic status worldwide. This neurologic disorder is caused primarily by the specific deterioration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNc). Unfortunately, there are no therapeutic agents that slow or delay the disease progression. Herein, menstrual stromal cell-derived dopamine-like neurons (DALNs) intoxicated with paraquat (PQ2+)/maneb (MB) were used as a model system to elucidate the mechanism by which CBD protects the neural cell from apoptosis in vitro. According to immunofluorescence microscopy, flow cytometry, cell-free assay, and molecular docking analysis, we demonstrate that CBD offers protection to DALNs against PQ2+ (1 mM)/MB (50 μM)-induced oxidative stress (OS) by simultaneously (i) decreasing reactive oxygen species (ROS: O2•-, H2O2), (ii) maintaining the mitochondrial membrane potential (ΔΨm), (iii) directly binding to stress sensor protein DJ-1, thereby blunting its oxidation from DJ-1CYS106-SH into DJ-1CYS106-SO3, and (iv) directly binding to pro-apoptotic protease protein caspase 3 (CASP3), thereby disengaging neuronal dismantling. Furthermore, the protective effect of CBD on DJ-1 and CASP3 was independent of CB1 and CB2 receptor signaling. CBD also re-established the Ca2+ influx in DALNs as a response to dopamine (DA) stimuli under PQ2+/MB exposure. Because of its powerful antioxidant and antiapoptotic effects, CBD offers potential therapeutic utility in the treatment of PD.

Advanced network pharmacology study reveals multi-pathway and multi-gene regulatory molecular mechanism of Bacopa monnieri in liver cancer based on data mining, molecular modeling, and microarray data analysis

Liver cancer is a malignant tumor that grows on the surface or inside the liver. The leading cause is a viral infection with hepatitis B or C virus. Natural products and their structural analogues have historically made a major contribution to pharmacotherapy, especially for cancer. A list of studies evidences the therapeutic efficacy of Bacopa monnieri against liver cancer, but the precise molecular mechanism is yet to be discovered. This study combines data mining, network pharmacology, and molecular docking analysis to potentially revolutionize liver cancer treatment by identifying effective phytochemicals. Initially, the information on active constituents of B. monnieri and target genes of both liver cancer and B. monnieri were retrieved from literature as well as from publicly available databases. Based on the matching results between B. monnieri potential targets and liver cancer targets, the protein-protein interaction (PPI) network was constructed using the STRING database and imported into Cytoscape for screening of hub genes based on their degree of connectivity. Later, the interactions network between compounds and overlapping genes was constructed using Cytoscape software to analyze the network pharmacological prospective effects of B. monnieri on liver cancer. Gene Ontology (GO) and KEGG pathway analysis of hub genes revealed that these genes are involved in the cancer-related pathway. Lastly, the expression level of core targets was analyzed using microarray data (GSE39791, GSE76427, GSE22058, GSE87630, and GSE112790). Further, the GEPIA server and PyRx software were used for survival and molecular docking analysis, respectively. In summary, we proposed that quercetin, luteolin, apigenin, catechin, epicatechin, stigmasterol, beta-sitosterol, celastrol, and betulic acid inhibit tumor growth by affecting tumor protein 53 (TP53), interleukin 6 (IL6), RAC-alpha serine/threonine protein kinases 1 (AKT1), caspase-3 (CASP3), tumor necrosis factor (TNF), jun proto-oncogene (JUN), heat shot protein 90 AA1 (HSP90AA1), vascular endothelial growth factor A (VEGFA), epidermal growth factor receptor (EGFR), and SRC proto-oncogene (SRC). Through, microarray data analysis, the expression level of JUN and IL6 were found to be upregulated while the expression level of HSP90AA1 was found to be downregulated. Kaplan–Meier survival analysis indicated that HSP90AA1 and JUN are promising candidate genes that can serve as diagnostic and prognostic biomarkers for liver cancer. Moreover, the molecular docking and molecular dynamic simulation of 60ns well complemented the binding affinity of the compound and revealed strong stability of predicted compounds at the docked site. Calculation of binding free energies using MMPBSA and MMGBSA validated the strong binding affinity between the compound and binding pockets of HSP90AA1 and JUN. Despite that, in vivo and in vitro studies are mandatory to unveil pharmacokinetics and biosafety profiles to completely track the candidature status of B. monnieri in liver cancer.

Allosteric Tuning of Caspase-7: Establishing the Nexus of Structure and Catalytic Power.

Caspase-7 (C7), a cysteine protease involved in apoptosis, is a valuable drug target for its role in human diseases (e.g. Parkinson's, Alzheimer's, sepsis). The C7 allosteric site has great potential for small molecule targeting, but numerous drug discovery efforts have identified precious few allosteric inhibitors. Here we present the first selective, drug-like inhibitor of C7 along with several other improved inhibitors based on our previous fragment hit. We also provide a rational basis for the impact of allosteric binding on the C7 catalytic cycle using an integrated approach including X-ray crystallography, stopped-flow kinetics, and molecular dynamics simulations. Our findings suggest allosteric binding disrupts C7 pre-acylation via neutralization of the catalytic dyad, displacement of substrate from the oxyanion hole, and altered dynamics of substrate binding loops. This work advances drug targeting efforts and bolsters our understanding of allosteric structure activity relationships (ASARs).

Oxidative Stress, Inflammatory, Angiogenic, and Apoptotic molecules in Proliferative Diabetic Retinopathy and Diabetic Macular Edema Patients.

The aim of this study is to evaluate molecules involved in oxidative stress (OS), inflammation, angiogenesis, and apoptosis, and discern which of these are more likely to be implicated in proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME) by investigating the correlation between them in the plasma (PLS) and vitreous body (VIT), as well as examining data obtained from ophthalmological examinations. Type 2 diabetic (T2DM) patients with PDR/DME (PDRG/DMEG; n = 112) and non-DM subjects as the surrogate controls (SCG n = 48) were selected according to the inclusion/exclusion criteria and programming for vitrectomy, either due to having PDR/DME or macular hole (MH)/epiretinal membrane (ERM)/rhegmatogenous retinal detachment. Blood samples were collected and processed to determine the glycemic profile, total cholesterol, and C reactive protein, as well as the malondialdehyde (MDA), 4-hydroxynonenal (4HNE), superoxide dismutase (SOD), and catalase (CAT) levels and total antioxidant capacity (TAC). In addition, interleukin 6 (IL6), vascular endothelial growth factor (VEGF), and caspase 3 (CAS3) were assayed. The VITs were collected and processed to measure the expression levels of all the abovementioned molecules. Statistical analyses were conducted using the R Core Team (2022) program, including group comparisons and correlation analyses. Compared with the SCG, our findings support the presence of molecules involved in OS, inflammation, angiogenesis, and apoptosis in the PLS and VIT samples from T2DM. In PLS from PDRG, there was a decrease in the antioxidant load (p < 0.001) and an increase in pro-angiogenic molecules (p < 0.001), but an increase in pro-oxidants (p < 0.001) and a decline in antioxidants (p < 0.001) intravitreally. In PLS from DMEG, pro-oxidants and pro-inflammatory molecules were augmented (p < 0.001) and the antioxidant capacity diminished (p < 0.001), but the pro-oxidants increased (p < 0.001) and antioxidants decreased (p < 0.001) intravitreally. Furthermore, we found a positive correlation between the PLS-CAT and the VIT-SOD levels (rho = 0.5; p < 0.01) in PDRG, and a negative correlation between the PSD-4HNE and the VIT-TAC levels (rho = 0.5; p < 0.01) in DMEG. Integrative data of retinal imaging variables showed a positive correlation between the central subfield foveal thickness (CSFT) and the VIT-SOD levels (rho = 0.5; p < 0.01), and a negative correlation between the CSFT and the VIT-4HNE levels (rho = 0.4; p < 0.01) in PDRG. In DMEG, the CSFT displayed a negative correlation with the VIT-CAT (rho = 0.5; p < 0.01). Exploring the relationship of the abovementioned potential biomarkers between PLS and VIT may help detecting early molecular changes in PDR/DME, which can be used to identify patients at high risk of progression, as well as to monitor therapeutic outcomes in the diabetic retina.