Caspase-3 Protein Research Articles

The Effect of Sildenafil Citrate (Viagra®) on Nasal Olfactory Mucosa of Adult Male Albino Rat: caspase-3 and inducible nitric oxide synthase protein expressions.

The Effect of Sildenafil Citrate (Viagra®) on Nasal Olfactory Mucosa of Adult Male Albino Rat: caspase-3 and inducible nitric oxide synthase protein expressions.

Long-term Cu exposure alters CYP450s activity and induces jejunum injury and apoptosis in broilers.

Copper (Cu) is an essential trace element that plays a crucial role in numerous physiopathological processes related to human and animal health. In the poultry industry, Cu is used to promote growth as a feed supplement, but excessive use can lead to toxicity on animals. Cytochrome P450 enzymes (CYP450s) are a superfamily of proteins that require heme as a cofactor and are essential for the metabolism of xenobiotic compounds. The purpose of this study was to explore the influence of exposure to Cu on CYP450s activity and apoptosis in the jejunum of broilers. Hence, we first simulated the Cu exposure model by feeding chickens diets containing different amounts of Cu. In the present study, histopathological observations have revealed morphological damage to the jejunum. The expression levels of genes and proteins of intestinal barrier markers were prominently downregulated. While the mRNA expression level of the gene associated with CYP450s was significantly increased. Additionally, apoptosis-related genes and proteins (Bak1, Bax, Caspase-9, Caspase-3, and CytC) were also significantly augmented by excessive Cu, while simultaneously decreasing the expression of Bcl-2. It can be concluded that long-term Cu exposure affects CYP450s activity, disrupts intestinal barrier function, and causes apoptosis in broilers that ultimately leads to jejunum damage.

Density Functional Theory-Guided Photo-Triggered Anticancer Activity of Curcumin-Based Zinc(II) Complexes.

Photodynamic therapy (PDT) has evolved as a new therapeutic modality for cancer treatment with fewer side effects and drug resistance. Curcumin exhibits PDT activity, but its low bioavailability restricts its clinical application. Here, the bioavailability of curcumin was increased by its complex formation with the Zn(II) center. For a structure-activity relationship study, Zn(II)-based complexes (1-3) comprising N^N-based ligands (2,2'-bipyridine in 1 and 2 or 1,10-phenanthroline in 3) and O^O-based ligands (acetylacetone in 1, monoanionic curcumin in 2 and 3) were synthesized and thoroughly characterized. The X-ray structure of the control complex, 1, indicated a square pyramidal shape of the molecules. Photophysical and TD-DFT studies indicated the potential of 2 and 3 as good visible light type-II photosensitizers for PDT. Guided by the TD-DFT studies, the low-energy visible light-triggered singlet oxygen (1O2) generation efficacy of 2 and 3 was explored in solution and in cancer cells. As predicted by the TD-DFT calculations, these complexes produced 1O2 efficiently in the cytosol of MCF-7 cancer cells and ultimately displayed excellent apoptotic anticancer activity in the presence of light. Moreover, the molecular docking investigation showed that complexes 2 and 3 have very good binding affinities with caspase-9 and p-53 proteins and could activate them for cellular apoptosis. Further molecular dynamics simulations confirmed the stability of 3 in the caspase-9 protein binding site.

MiR-200a involvement in the biological behavior of hepatoma carcinoma cells by targeting the regulatory expression of mesenchymal-epithelial transition factor

Objective: To study the regulatory effect of miR-200a on mesenchymal-epithelial transition factor (MET) and its impact on the biological behavior of hepatoma carcinoma cells. Method: A luciferase reporter assay was used to determine miR-200a's regulatory impact on MET. Human hepatoma HepG2 cells were divided into a control group, a miR-200a group, a MET overexpression group, and a co-transfection group (miR-200a+MET). After culture, cell proliferation ability, cell migration ability, apoptosis, cell invasion ability, and the expression of MET and apoptosis-related (Bcl-2, Caspase-3, Bax) proteins were detected and observed by cell counting kit-8 (CCK-8), scratch assay, Annexin V-FITC staining, transwell chambers, and western blotting. The two groups were compared using the independent sample t-test. The multiple groups were statistically analyzed using one-way ANOVA. Results: The luciferase experiment showed that miR-200a had target MET. The proliferation rate, number of invasions in cells (55.00 ± 7.21, 85.00 ± 7.94, 164.67 ± 19.22, 104.00± 12.29), scratch healing rate (28.33% ± 5.03%, 61.67% ± 4.04%, 74.67% ± 7.02%, 49.33% ± 9.02%), and expression levels of MET, Bcl-2, and Caspase-3 proteins were lower in the miR-200a group than those in the control group, MET overexpression group, and co-transfection group, while the MET overexpression group had higher indexes than the other three groups, with statistically significant differences between the groups (P <0.05). The apoptosis rate of HepG2 cells and the expression level of Bax protein were higher in the miR-200a group than those in the control group, MET overexpression group, and co-transfection group (19.25% ± 2.98%, 6.80% ± 1.15%, 3.42% ±0.76%, 9.90% ± 2.72%), while the levels of various indexes in the MIF overexpression group were lower than those in the other three groups. The control group and co-transfection group were between the two groups, and the difference between the groups was statistically significant (P <0.05). Conclusion: HepG2 cell proliferation, migration, invasion, and cell apoptosis induction can be inhibited by miR-200a, and the functional mechanism for this may be associated with the miR-200a target's ability to down-regulate MET expression in HepG2 cells.

L-valine supplementation disturbs vital molecular pathways and induces apoptosis in mouse testes

The branched-chain amino acids (BCAAs: leucine, isoleucine and valine) are essential for animal growth and metabolic health. However, the effect of valine on male reproduction and its underlying molecular mechanism remain largely unknown. Here, we showed that l-valine supplementation (0.30% or 0.45%, water drinking for 3 weeks) did not change body and testis weights, but significantly altered morphology of sertoli cells and germ cells within seminiferous tubule, and enlarged the space between seminiferous tubules within mouse testis. l-valine treatment (0.45%) increased significantly the Caspase3/9 mRNA levels and CASPASE9 protein levels, therefore induced apoptosis of mouse testis. Moreover, gene expression levels related to autophagy (Atg5 and Lamb3), DNA 5 mC methylation (Dnmt1, Dnmt3a, Tet2 and Tet3), RNA m6A methylation (Mettl14, Alkbh5 and Fto), and m6A methylation binding proteins (Ythdf1/2/3 and Igf2bp1/2) were significantly reduced. Protein abundances of ALKBH5, FTO and YTHDF3 were also significantly reduced, but not for ATG5 and TET2. Testis transcriptome sequencing detected 537 differentially expressed genes (DEGs, 26 up-regulated and 511 down-regulated), involved in multiple important signaling pathways. RT-qPCR validated 8 of 9 DEGs (Cd36, Scd1, Insl3, Anxa5, Lcn2, Hsd17b3, Cyp11a1, Cyp17a1 and Agt) to be decreased significantly, consistent with RNA-seq results. Taken together, l-valine treatment could disturb multiple signaling pathways (autophagy and RNA methylation etc.), and induce apoptosis to destroy the tissue structure of mouse testis.

Gypenoside XVII attenuates renal ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress and NLRP3 inflammasome-triggered pyroptosis

BackgroundRenal ischemia-reperfusion (I/R) is one of the main causes of acute kidney injury (AKI), for which there is currently no effective treatment. Recently, the interaction between endoplasmic reticulum (ER) stress and pyroptosis during AKI has been investigated. AimThe purpose of this study was to investigate the protective effects of Gypenoside XVII (GP-17) against I/R-induced renal injury. MethodsIn this study, mice were divided into 6 groups, sham group, I/R group, GP-17 low-, medium-, high-dose group, and positive control 4-PBA group. The renal I/R was performed in mice by clamping the bilateral renal pedicles for 40 min, and then reperfusing for 24 h. Blood and kidney samples were collected for analysis. ResultsThe results showed that GP-17 improved renal function and alleviated renal histopathological abnormalities caused by I/R. In addition, GP-17 pretreatment significantly decreased the expression or phosphorylation of ER stress response proteins including BIP, p-PERK, and CHOP. Besides, GP-17 inhibited the expression of pyroptosis proteins including caspase-1, GSDMD, and apoptotic protein BAX. The inflammatory factor IL-1β in these GP-17 pretreatment groups was also significantly reduced. ConclusionGP-17 blocked NLRP3 inflammasome activation by inhibiting ERS, thereby inhibiting renal tubular cell pyroptosis and apoptosis, and prevented renal I/R injury.

A novel photosensitizer DTPP-mediated photodynamic therapy induces oxidative stress and apoptosis through mitochondrial pathways in LA795 cells

ObjectiveInvestigation of the effects of 5–5- (4-N, N-diacetoxylphenyl)-10,15,20- tetraphenylporphyrin (DTPP)-mediated photodynamic therapy (PDT) on oxidative stress and mitochondrial apoptosis in LA795 lung cancer cells. MethodsProteomics was used to identify differentially expressed proteins after PDT treatment. The apoptosis rate was determined by flow cytometry. Morphologic observation of apoptosis, reactive oxygen species (ROS) levels, antioxidant indices, nitric oxide (NO) content, mitochondrial membrane potential (MMP), and Caspase- 9 and Caspase-3 were determined by assays; apoptosis-related protein levels of Cytochrome (Cyto) c, Bcl- 2, Bax were determined by Western blot. ResultsTypical apoptosis morphology of LA795 cells was observed after PDT. The cells were mainly in the apoptosis death pathway with high cell apoptosis rates. The proteomics study observed the apoptosis-associated proteins, oxidative stress proteins, antioxidant proteins, the cytoskeletal protein and mitochondrial dysfunction in LA 795 cells. Additional results indicated that PDT could increase levels of ROS, NO; decrease glutathione (GSH) content and MMP; upregulated Bax, Cyto c, and Caspase-3 protein expression, inhibited Bcl-2 protein expression, and further induced cell apoptosis. The effect of DTPP-PDT on lung cancer was: first, mitochondrial Cyto c is released into the cytoplasm, then Caspase- 9 / Caspase-3 was activated, Bcl-2 decreased/Bax increased, initiating cell apoptosis. ConclusionDTPP-PDT could induce oxidative stress and apoptosis via mitochondrial pathways in LA795 cells.

Estimating the in vitro cytotoxicity of the newly emerged zinc oxide (ZnO) doped chromium nanoparticles using the human fetal lung fibroblast cells (WI38 cells)

Advances in nanotechnology have been increased for more smart applications and getting the highest level of benefits, recently modification of the surface characters of nanoparticles is a new trend to get the optimal benefits, one of these modification is doping of zinc oxide with chromium nanoparticles (ZnO doped Cr NPs), the present study aimed to identify the surface characters of doped ZnO and their possible cytotoxic effects. The doped NPs were characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR), Field emission scanning electron microscope (FESEM), and Electromagnetic Data Exchange (EDX). Human fetal lung fibroblast cells (WI38 Cells) was treated with variable concentrations of pure ZnO and ZnO doped Cr (0.01 %, 0.02 %, 0.03 % and 0.04 %) for 24 hr at 37 °C followed by the MTT assay. The cells treated with the obtained half-maximal inhibitory concentration (IC50). The supernatant and cells were collected for oxidant/anti-oxidant and molecular analysis.The observed FESEM features are in line with the reported XRD analysis confirming the hexagonal crystal symmetry of all samples. The findings revealed that pure ZnO exhibited potent cytotoxic effects followed by (0.03 % and 0.04 %). All tested NPs produce lipid peroxidation significantly (0.03 % and 0.04 %). The significant up regulation of Bcl-2-associated X protein (BAX) and apoptotic Caspase (Cas-3) transcription level were reported in ZnO and 0.03 % and 0.04 % in contrast the anti apoptitic B-cell lymphoma 2 (Bcl-2) is elevated in 0.01 % and 0.02 %. Doping of ZnO with Cr causing significant morphological changes which effect on their toxicity especially with 0.03 % and 0.04 %.

Design, synthesis of novel chromene-based scaffolds targeting hepatocellular carcinoma: Cell cycle arrest, cytotoxic effect against resistant cancer cells, apoptosis induction, and c-Src inhibition.

New chromene derivatives were synthesized based on 4-(3,4-dimethoxy)-4H-chromene scaffold. All target compounds exhibited cytotoxic activity against HepG2 cells (IC50 = 2.40-141.22 μM). Chromens 5 and 9 showed superior cytotoxicity over staurosporine (IC50 = 18.27 μM) and vinblastine (IC50 = 5.20 μM). c-Src kinase inhibition assay of compounds 5 and 9 displayed the dominant c-Src inhibitory activity of 5 (IC50 = 0.184 μM) over 9 (IC50 = 0.288 μM). The safety of the most potent compound 5 against normal WI-38 cells was confirmed via its IC50 of 115.75 μM comparable with 5-FU (IC50 = 16.28 μM). Moreover, the promising chromene 5 displayed potent cytotoxicity against resistant HepG2 cells with IC50 of 26.03 μM comparable with 5-FU (IC50 = 42.68 μM). The most active chromene 5 arrested the HepG2 cell cycle at the S phase and induced a 29-fold increase in the total number of apoptotic cells indicating pre-G1 apoptosis. The ability of compound 5 to induce apoptosis was supported via elevation of caspase-3, caspase-7, caspase-9 and proapoptotic Bax protein levels in addition to downregulation of the antiapoptotic Bcl2 protein. Molecular docking studies of compound 5 showed good binding interaction pattern inside c-Src kinase enzyme active site.

Anti-tumor effect of Fasudil, a ROCK inhibitor, on gastric cancer in mice

Gastric cancer remains one of the most common malignant tumors and a leading cause of death. However, there are few reports about the anti-tumor effect of Fasudil (Fas) on the stomach. Establishment of nude mice model of gastric cancer to evaluate the anti-tumor effect of Fas. The mice in each group were weighed every week. Serum and tumor tissue cytokines are detected by commercial kits. Serum and tumor tissue superoxide dismutase and malondialdehyde were detected by commercial kits. Histopathological changes of the tumor were detected by HE staining. Expression of ROS/ TXNIP/NLRP3/Caspase-1 pathway in tumor tissues was detected by Western blot. Our results showed that Fas increased and decreased the body weight of tumor mice, increased serum and tumor tissue cytokines contents of tumor mice, increased serum and tumor tissue oxidative stress of tumor mice, and increased Bax, Caspase-3, Caspase-9 protein expression, reduced Bcl-2 protein expression, also regulated ROS/TXNIP/NLRP3/Caspase-1 pathway expression. Our experiments show that Fas has a significant anti-gastric cancer effect, and its mechanism is related to the regulation of oxidative stress, inflammation and apoptosis in vivo.

Inhibition of Sema4D attenuates pressure overload-induced pathological myocardial hypertrophy via the MAPK/NF-κB/NLRP3 pathways

Sema4D (CD100) is closely related to pathological and physiological processes, including tumor growth, angiogenesis and cardiac development. Nevertheless, the role and mechanism of Sema4D in cardiac hypertrophy are still unclear to date. To assess the impact of Sema4D on pathological cardiac hypertrophy, TAC surgery was performed on C57BL/6 mice which were transfected with AAV9-mSema4D-shRNA or AAV9-mSema4D adeno-associated virus by tail vein injection. Our results indicated that Sema4D knockdown mitigated cardiac hypertrophy, fibrosis and dysfunction when exposed to pressure overload, and Sema4D downregulation markedly inhibited cardiomyocyte hypertrophy induced by angiotensin II. Meanwhile, Sema4D overexpression had the opposite effect in vitro and in vivo. Furthermore, analysis of signaling pathways showed that Sema4D activated the MAPK pathway during cardiac hypertrophy induced by pressure overload, and the pharmacological mitogen-activated protein kinase kinase 1/2 inhibitor U0126 almost completely reversed Sema4D overexpression-induced deteriorated phenotype, resulting in improved cardiac function. Further research indicated that myocardial hypertrophy induced by Sema4D was closely related to the expression of the pyroptosis-related proteins PP65, NLRP3, caspase-1, ASC, GSDMD, IL-18 and IL-1β. In conclusion, our study demonstrated that Sema4D regulated the process of pathological myocardial hypertrophy through modulating MAPK/NF-κB/NLRP3 pathway, and Sema4D may be the promising interventional target of cardiac hypertrophy and heart failure.

Porcupine quills keratin peptides induces G0/G1 cell cycle arrest and apoptosis via p53/p21 pathway and caspase cascade reaction in MCF-7 breast cancer cells.

Porcupine quills, a by-product of porcupine pork, are rich in keratin, which is an excellent source of bioactive peptides. The objective of this study was to investigate the underlying mechanism of anti-proliferation effect of porcupine quills keratin peptides (PQKPs) on MCF-7 cells. Results showed that PQKPs induced MCF-7 cells apoptosis by significantly decreasing the secretion level of anti-apoptosis protein Bcl-2 and increasing the secretion levels of pro-apoptosis proteins Bax, cytochrome c, caspase 9, caspase 3 and PARP. PQKPs also arrested the cell cycle at G0/G1 phase via remarkably reducing the protein levels of CDK4 and enhancing the protein levels of p53 and p21. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis identified nine peptides with molecular weights less than 1000 Da in PQKPs. Molecular docking results showed that TPGPPT and KGPAC identified from PQKPs could bind with p53 mutant and Bcl-2 protein by conventional hydrogen bonds, carbon hydrogen bonds and van der Waals force. Furthermore, the anti-proliferation impact of synthesized peptides (TPGPPT and KGPAC) was shown in MCF-7 cells. These findings indicated that PQKPs suppressed the proliferation of MCF-7 breast cancer cells by triggering apoptosis and G0/G1 cell cycle arrest. Moreover, the outcome of this study will bring fresh insights into the production and application of animal byproducts. © 2023 Society of Chemical Industry.

ASC/inflammasome-independent pyroptosis in ovarian cancer cells through translational augmentation of caspase-1

Canonical pyroptosis is type of programmed cell death depending on active caspase-1, and the inflammasome carries out caspase-1 activation. Here, we showed that docosahexaenoic acid (DHA) induced ovarian cancer cell deaths in caspase-1-dependent manner. DHA increased caspase-1 activity and led to interleukin-1β secretion and gasdermin D cleavage while disulfiram inhibited DHA-induced cell death, suggesting that DHA triggered pyroptosis. Intriguingly, ASC, the molecule recruiting caspase-1 to inflammasome for activation, was dispensable for DHA-induced pyroptosis. Instead, we observed remarkable elevation in caspase-1 abundance concurrent with the activation of caspase-1 in DHA-treated cells. As ectopically overexpressing caspase-1 resulted in robust amount of active caspase-1, we reason that DHA activates caspase-1 and pyroptosis through the generation of excessive amount of caspase-1 protein. Mechanistically, DHA increased caspase-1 by specifically accelerating caspase-1 protein synthesis via the p38MAPK/Mnk1 signaling pathway. We have uncovered an unknown pyroptosis mechanism in which caspase-1-dependent pyroptosis can occur without the participation of ASC/inflammasome.

Design, synthesis, and evaluation of n-butylphthalide and ligustrazine hybrids as potent neuroprotective agents for the treatment of ischemic stroke in vitro and in vivo

A series of novel NBP-TMP hybrids with neuroprotective effects were designed and synthesized for the treatment of ischemic stroke. The anti-cerebral ischemic activity of these compounds was screened by evaluating their neuroprotective effects on the oxygen glucose deprivation/reperfusion (OGD/R)-induced SH-SY5Y cell injury model in vitro. Nine compounds 7e, 7h-7i, 7k, 7m-7p and 7r showed better activities on cell viability and LDH levels compared to NBP at the concentration of 6.25 μM. Among them, compound 7m showed the best potency with a percentage of protection 90.2 % compared to NBP (69.2 %) and other compounds. Preliminary structure-activity analysis revealed that the introduction of iodine and N-methylpiperazine groups could significantly improve the neuroprotective effect. Further mechanism research showed that compound 7m could reduce the damage to neuronal mitochondria caused by OGD/R by reducing ROS and increasing mitochondrial membrane potential (MMP), and reduce the apoptosis and necrosis of neurons to play a neuroprotective role. In addition, 7m could regulate the levels of mitochondrial apoptosis pathway-related proteins Bcl-2, Bax, and caspase 3. Finally, in vivo experiments showed that the compound 7m significantly inhibited ischemia-reperfusion injury and cerebral blood flow in rats, and showed a more significant neuroprotective effect than the positive drug NBP at a dose concentration of 20 mg/kg. In conclusion, our results suggest that 7m may be used as a novel lead compound for the future development of anti-cerebral ischemic agents.

Mechanism of DaiTongXiao in the treatment of gouty arthritis through the NLRP3 signaling pathway

Ethnopharmacological relevanceDaiTongXiao (DTX) is a traditional Chinese Dai folk formulation utilized for gouty arthritis treatment, with substantial evidence supporting its anti-inflammatory properties. The NLRP3 inflammasome disorder is tightly linked to the development of many inflammatory diseases. Aim of the studyTo elucidate the therapeutic efficacy of DTX in gouty arthritis and reveal its potential underlying mechanism. Materials and methodsThe primary active constituents in DTX were determined through ultraviolet spectrophotometry and gas chromatography. Rats underwent induction with monosodium urate (MSU), followed by treatment of J774A.1 cells with adenosine triphosphate (ATP) activation and lipopolysaccharide (LPS) induction and the subsequent culture in Dulbecco’s modified Eagle’s medium. The degree of foot joint swelling in rats was assessed, and ankle joints were evaluated through H&E staining. Enzyme-linked immunosorbent assay was performed to measure the levels of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α in both serum and cells. Reverse transcription–polymerase chain reaction (RT-PCR) was performed to determine the relative mRNA expression levels of NLRP3, ASC, Caspase-1, and NF-κB in J774A.1 macrophages. The expression of NLRP3, ASC, Caspase-1, and NF-κB was examined by western blotting. ResultsDTX could alleviate MSU-induced joint swelling in rats, as evidenced by a reduction in joint inflammation. Moreover, DTX effectively enhanced the survival rate of J774A.1 cells following LPS induction and ATP activation. Furthermore, DTX significantly reduced IL-1β, IL-6, IL-8, and TNF-α levels in both cell culture medium and rat serum. RT-PCR results revealed that DTX notably downregulated the mRNA expression levels of NLRP3, ASC, Caspase-1, and NF-κB in J774A.1 cells. Additionally, DTX downregulated NLRP3, ASC, NF-κB, and Caspase-1 expression in the joint tissue. ConclusionsDTX exerts a significant anti–gouty arthritis effect, with its mechanism being tightly linked to the NLRP3 inflammatory signaling pathway. This pathway may be modulated by inhibiting IL-1β differentiation and maturation by downregulating NLRP3, ASC, Caspase-1, and NF-κB protein expression. This, in turn, leads to a reduction in the release of IL-6, IL-8, and TNF-α, ultimately impeding gouty arthritis progression.

(E)-2-Methoxy-4-(3-(4-Methoxyphenyl) Prop-1-en-1-yl) Phenol Suppresses Breast Cancer Progression by Dual-Regulating VEGFR2 and PPARγ.

In cancer treatment, multi-target approach has paid attention to a reasonable strategy for the potential agents. We investigated whether (E)-2-methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) phenol (MMPP) could exert an anticancer effect by dual-regulating VEGFR2 and PPARγ. MMPP showed modulating effects in TNBC type (MDA-MB-231 and MDA-MB-468) and luminal A type (MCF7) breast cancer cell lines. MMPP enhanced PPARγ transcriptional activity and inhibited VEGFR2 phosphorylation. MMPP-induced signaling by VEGFR2 and PPARγ ultimately triggered the downregulation of AKT activity. MMPP exhibited anticancer effects, as evidenced by growth inhibition, inducement of apoptosis, and suppression of migration and invasion. At the molecular level, MMPP activated pro-apoptotic proteins (caspase3, caspase8, caspase9, and bax), while inhibiting the anti-apoptotic proteins (bcl2). Additionally, MMPP inhibited the mRNA expressions of EMT-promoting transcription factors. Therefore, our findings showed molecular mechanisms of MMPP by regulating VEGFR2 and PPARγ, and suggested that MMPP has potential to treat breast cancer.

Fatty acid modification of casein bioactive peptides nano-assemblies, synthesis, characterization and anticarcinogenic effect

In this study, the nano-assemblies of bovine casein hydrolyzed peptides (HP) modified by fatty acids with various alkyl chain lengths (C8, C10, C12 and C14) were synthesized. The physicochemical properties of HP-C8-HP-C14 nano-assemblies were characterized using spectra, laser particle size analyzer, contact angle meter, scanning electron microscope (SEM) and cryo-transmission electron microscope (Cryo-TEM). HP-C8 and HP-C10 self-assembled into a hollow cube cage with an average size of ~500 nm, and the assembly of HP-C12 showed a flower-shaped morphology with more dispersed behavior, and droplet size was observed as ~20 nm. The in vitro cytotoxicity against human breast cancer cells MCF-7 was tested using CCK-8 assay and flow cytometry analysis. HP-C12 showed the highest cytotoxicity for MCF-7 cells with an inhibition rate of 66.03 % ± 0.35 % with an IC50 value of 7.4 μM among HP-Cn. HP-C8, HP-C10 and HP-C12 significantly affected on the migration, invasion and apoptosis of MCF-7 cells. The apoptosis mechanism may depend on the upregulation of anti-apoptotic protein Bcl-2 as well as pro-apoptotic proteins Bax and caspase-8. The dead MCF-7 cells were analyzed with UHPLC-MS/MS using untargeted metabolomics, revealing key metabolic pathways.

Mechanisms of Inflammatory Caspase Activation in Sickle Cell Disease

Background: Sickle cell disease (SCD) is an inherited red blood cell disorder that impacts millions of people worldwide. Patients with SCD experience chronic hemolysis and have elevated serum levels of free heme. Free heme activates a variety of inflammatory pathways that contribute to the serious clinical complications of SCD. One of the inflammatory pathways activated by heme is the inflammatory caspase pathway. Our lab previously showed that heme exposure in macrophages leads to the activation of inflammatory caspase proteins caspase-1 and caspase-4. Activation of these proteins by heme leads to IL-1β release and pyroptosis, a form of inflammatory cell death. Patients with SCD have increased circulating IL-1β levels, indicating this pathway is active in patients. In mouse models, inflammatory caspases are required for hemolysis-induced neutrophil recruitment and lethality, indicating these proteins are central to hemolysis-induced inflammation. Despite the key role these proteins play following hemolysis, it remains unknown how heme activates inflammatory caspases. Interestingly, both caspase-1 and caspase-4 are required for heme-induced IL-1β release while caspase-4 alone is required for heme-induced pyroptosis. In fact, pyroptosis likely is required for IL-1β release from the cell. Based on this data, we hypothesized that caspase-4 is the apical step in this pathway and a key druggable target for controlling unchecked inflammation in SCD. Results: We first sought to determine if caspase-4 activation is upstream of caspase-1 activation. To assess this, we used an imaging-based caspase activation reporter assay. Using this system, we found that siRNA knock-down of caspase-4 in primary human macrophages significantly inhibited heme-induced caspase-1 activation (63.8% vs 26.3%, n =4, p = 0.015). This result shows that caspase-4 is upstream of heme-induced caspase-1 activation and confirmed caspase-4 activation is the initial step in this pathway. Next, we sought to determine how caspase-4 activation occurs. Other known activators of caspase-4 induce activation by directly binding to caspase-4 and inducing self-oligomerization. To determine if heme acts in a similar manner, we used a published heme-binding prediction algorithm called HeMoQuest. We found multiple predicted heme-binding sites in caspase-4. The dissociation constants for these binding sites are less than 1 µM, suggesting strong binding affinity. Importantly, there are no predicted heme binding sites in caspase-1, indicating caspase-4 is the only direct heme receptor in this pathway. To validate these binding predictions, we performed molecular docking experiments and confirmed there are multiple heme-binding sites in caspase-4. Specifically, we found two heme-binding regions in the p10 catalytic subunit of caspase-4 comprising 2-4 essential residues. We next confirmed that heme directly interacts with caspase-4 in vitro. Using a pull-down assay with heme conjugated to agarose beads, we observed heme bound to purified caspase-4 and to casapase-4 in macrophage cell-free extracts. Heme did not interact with proteins structurally related to caspase-4 including caspase-1, caspase-7, and caspase-9 and caspase-4 was not pulled down by control agarose. This confirms the specificity of the heme-caspase-4 interaction. To determine if heme binding to caspase-4 results in activation, we performed native PAGE analysis of purified caspase-4 after exposure to heme. Heme induced formation of a caspase-4 oligomer around 200 kDa in size, four-fold larger than the inactive caspase-4 monomer. Other compounds known to induce IL-1β release from cells, like microbial toxin nigericin, did not induce caspase-4 oligomerization, demonstrating the specificity of heme's effect on caspase-4. Conclusions: We have determined that caspase-4 is a direct intracellular heme receptor and that heme exposure leads to caspase-4 activation, which in turn facilitates caspase-1 activation, IL-1β release and cell death. Our future work will elucidate the functional effects of heme binding to caspase-4 in mouse models of sickle cell disease. Caspase-4 is an attractive therapeutic target as it is required for both functional outputs of inflammatory caspase activation, IL-1β release and pyroptosis. Inhibiting heme-caspase-4 interactions would provide a specific way to block the harmful effects of this pathway in patients.

Delineating involvement of MAPK/NF-κB pathway during mitigation of permethrin-induced oxidative damage in fish gills by melatonin

Present study evaluated involvement of transcription factors during permethrin-induced gill toxicity and its amelioration by melatonin. First, adult Notoptertus notopterus females were exposed to permethrin at nominal concentrations [C: 0.0, P1: 0.34, P2: 0.68 µg/L] for 15 days followed by intramuscular melatonin administration (100 µg/kg body weight) for 7 days. Gill MDA, XO, LDH levels increased, while Na+-K+-ATPase, SDH, cytochrome C oxidase levels decreased with increasing permethrin concentrations. Glutathione, SOD, CAT, GST, GRd levels increased in P1 than C, but decreased in P2 than P1, C. Melatonin administration restored gill enzyme and antioxidant levels in P1, P2. Next, isolated gill tissues were exposed to permethrin at 25, 50 µM doses along with melatonin administration (100 μg/mL). NF-κB, NRF2, Keap1, ERK, Akt, caspases protein expression changed significantly during permethrin-induced gill damage. Melatonin administration amended permethrin-induced molecular imbalance through modulation of caspase proteins and MAPK/NF-κB signal transduction pathway via melatonin receptor 1.

Increasing Apoptotic Effect of Cord Blood and Wharton's Jelly-derived Mesenchymal Stem Cells on HT-29.

Colorectal cancer (CRC) is the third most common cancer worldwide. Recently, mesenchymal stem cells (MSCs) have been considered a suitable cell therapy option for cancer due to their high migration rate to the tumor site. The study aimed to compare the effects of human umbilical cord blood derived-MSC (UCMSC) and human Wharton's Jelly derived-MSC (WJ-MSC) on the HT-29 cell line. UC-MSC was obtained by Ficoll-Paque density gradient and WJ-MSC by explant method. The characterizations of MSCs and apoptosis assays were performed by flow cytometry, and caspase-3 protein levels were measured by ELISA. After 72 hours of HT-29 cancer cells incubation, it was indicated that WJ-MSC was more effective at 1:5 and 1:10 ratios. Similar results were found for caspase-3 by ELISA. Moreover, WJ-MSC (1:5, p < 0.006; 1:10, p < 0.007) was found to be more effective at both doses compared to UC-MSC. In this study, we used two different MSC sources at two different ratios to evaluate the apoptotic effect of MSC in vitro on HT-29 CRC cells. As a result, WJ-MSC indicated a more apoptotic effect on HT-29 cells compared to CB-MSC. We anticipated that this preliminary in vitro study would be extended in future in vitro/in vivo studies. Moreover, investigating the behavior of MSC in colorectal tumor microenvironment will be beneficial for the stem cell therapy approach.