Caspase-3 Activity Research Articles

Autism risk gene Cul3 alters neuronal morphology via caspase-3 activity in mouse hippocampal neurons.

Autism Spectrum Disorders (ASDs) are neurodevelopmental disorders (NDDs) in which children display differences in social interaction/communication and repetitive stereotyped behaviors along with variable associated features. Cul3, a gene linked to ASD, encodes CUL3 (CULLIN-3), a protein that serves as a key component of a ubiquitin ligase complex with unclear function in neurons. Cul3 homozygous deletion in mice is embryonic lethal; thus, we examine the role of Cul3 deletion in early synapse development and neuronal morphology in hippocampal primary neuronal cultures. Homozygous deletion of Cul3 significantly decreased dendritic complexity and dendritic length, as well as axon formation. Synaptic spine density significantly increased, mainly in thin and stubby spines along with decreased average spine volume in Cul3 knockouts. Both heterozygous and homozygous knockout of Cul3 caused significant reductions in the density and colocalization of gephyrin/vGAT puncta, providing evidence of decreased inhibitory synapse number, while excitatory synaptic puncta vGulT1/PSD95 density remained unchanged. Based on previous studies implicating elevated caspase-3 after Cul3 deletion, we demonstrated increased caspase-3 in our neuronal cultures and decreased neuronal cell viability. We then examined the efficacy of the caspase-3 inhibitor Z-DEVD-FMK to rescue the decrease in neuronal cell viability, demonstrating reversal of the cell viability phenotype with caspase-3 inhibition. Studies have also implicated caspase-3 in neuronal morphological changes. We found that caspase-3 inhibition largely reversed the dendrite, axon, and spine morphological changes along with the inhibitory synaptic puncta changes. Overall, these data provide additional evidence that Cul3 regulates the formation or maintenance of cell morphology, GABAergic synaptic puncta, and neuronal viability in developing hippocampal neurons in culture.

The Effect of κ-Carrageenan on Porcine Sperm Cryo-Survival.

κ-Carrageenan is a sulfated polysaccharide from red seaweed with substantial antioxidant activities. This study aimed to investigate the effect of κ-Carrageenan treatment on frozen-thawed (FT) porcine semen quality. Therefore, the spermatozoa were diluted and cryopreserved in a freezing extender supplemented with 0 (control), 0.2, 0.4, 0.6, and 0.8 mg/mL κ-Carrageenan. Sperm kinematics were assessed immediately after thawing (AT) and post-incubation for 120 min. The viability, acrosome integrity, lipid peroxidation, mitochondrial membrane potential (MMP), and intracellular caspase activity were measured AT. The results indicated that 0.2 mg/mL κ-Carrageenan increased total and progressive motility AT and post-incubation for 120 min (p < 0.05). Moreover, the viable sperm percentage and MMP after 0.2 mg/mL treatment were higher than those after control and other κ-Carrageenan concentration treatments. The proportion of acrosome-intact spermatozoa was significantly higher after 0.2 and 0.4 mg/mL κ-Carrageenan treatment than that after control and other κ-Carrageenan concentration treatments. The intracellular caspase activity was not significantly different among the experimental groups. However, the MDA concentration after 0.2 mg/mL κ-Carrageenan treatment was lower (p < 0.05) than that after the control treatment. Taken together, adding κ-Carrageenan to the porcine semen freezing extender improved the FT sperm quality mainly by influencing membrane stability and protecting against oxidative stress.

NREP, transcriptionally upregulated by HIF-1α, aggravates breast cancer cell growth and metastasis by promoting glycolysis

Breast cancer (BC) poses a great threat to women’s health. Neuronal regeneration related protein (NREP) is a multifunctional protein that is involved in embryonic development, regeneration, and human disease. However, the biological function of NREP in tumors is rarely reported and its role in BC remains unknown. Bioinformatics analysis showed that NREP is highly expressed and closely correlated with poor survival in BC patients. Under hypoxic conditions, NREP was upregulated in BC cells, and this promotion was reversed by hypoxia-inducible factor HIF-1α suppression. Luciferase reporter system and chromatin immunoprecipitation assays confirmed that HIF-1α directly binds to the promoter of NREP to increase the transcriptional activity of NREP. NREP suppression inhibited cell proliferation, arrested the cell cycle at the G1/S phase, and promoted apoptosis and caspase-3 activity in BC cells. Suppression of NREP decreased the tube formation ability of HUVECs. In addition, NREP downregulation showed an inhibition effect on cell migration, invasion, and EMT of BC cells. In NREP overexpressed cells, all these changes were reversed. In vivo, animal experiments also confirmed that NREP promotes BC tumor growth and metastasis. In addition, NREP promoted cellular glycolysis and enhanced the levels of glucose consumption, ATP, lactate production, and glucose transporters expression in NREP-overexpressed BC cells. In summary, our results demonstrated that NREP could be transcriptional activated by HIF-1α, which may aggravate BC tumor growth and metastasis by promoting cellular glycolysis. This result suggested that NREP may play an essential part in BC progression.

Linalool-zinc oxide nanocomposite controls Toxoplasma gondii infection through inhibiting inflammation, oxidative stress, and pathogenicity.

The present in vitro and in vivo study aimed to fabricate and characterize linalool-zinc oxide nanoparticles (Lin-ZNP) and evaluate their effectiveness against Toxoplasma gondii infection in terms of inflammation, oxidative stress, and pathogenicity. Lin-ZNP was synthesized using an ethanolic solution of polyvinyl alcohol. The anti-Toxoplasma and cytotoxicity activities of Lin-ZNP were investigated, along with its effects on nitric oxide (NO) production, caspase-3 activity, and pro-inflammatory genes. After treating T. gondii-infected mice with Lin-ZNP for 14 days, the number and size of tissue cysts, antioxidant potential, pro-inflammatory cytokines, and T. gondii pathogenicity-related genes were evaluated by real-time polymerase chain reaction and Western blot analysis. The Lin-ZNP composite showed a reduced tendency with an average size of 105 nm. Lin-ZNP significantly reduced the viability of tachyzoites. The obtained selectivity index higher than 10, indicating high specificity for parasites with low cytotoxicity to normal cells. The Lin-ZNP significantly (p < 0.05) increased the production of NO, caspase-3 activity, and the expression levels of pro-inflammatory genes. Lin-ZNP significantly (p < 0.001) decreased the size and number of tissue cysts and caused a significant reduction in the level of malondialdehyde and a considerable increase (p < 0.001) in antioxidant enzymes and their expression genes. Lin-ZNP significantly downregulated both mRNA and protein expression of the inflammation-related markers associated with the TLRs/NF-κB pathway. The expression levels of the T. gondii pathogenicity-related genes were significantly downregulated (p < 0.05). The recent survey indicated that Lin-ZNP manages T. gondii infection by its antioxidant activity and inhibiting the TLRs/NF-κB pathway without toxicity in mice.

Caki-1 Spheroids as a 3D Model for Studying Free Fatty Acid-Induced Proximal Tubule Lipotoxicity

Three-dimensional (3D) multicellular spheroid shows a biological complexity resembling native tissue in comparison to conventional monolayer cultures, making it a better model system to assess drug effcacy and cytotoxicity. The main aim of this study was to examine the usefulness of Caki-1, a human proximal tubule (PT) representative cell line, as a 3D model system for studying free fatty acid-induced PT lipotoxicity. Caki-1 spheroids were generated and maintained on ultra-low attachment plates and characterized regarding time-dependent changes in morphology and functionality. In optimal 3D culture conditions, Caki-1 cells formed well-defined large compact 3D spheroids with uniform morphology, good circularity, and increased diameter from days 2-10. There was no change when the spheroids were incubated with lipid-free bovine serum albumin (BSA, 0.5%) from day 6 to 10. Chronic exposure to palmitate (300 μM) resulted in dispersed and flattened spheroid morphology, with a larger number of dead cells in the peripheral layers and decreased spheroid core. Moreover, palmitate-treated spheroids showed a significant increase in caspase-3 activity at the external layers of the spheroids, suggesting that the detached cells underwent apoptosis. Simultaneous addition of oleate (200 μM) prevents palmitate-induced spheroid disintegration and cell death in Caki-1 3D culture. Mechanistically, palmitate triggered endoplasmic reticulum (ER) stress and autophagy dysfunction, as evidenced by significantly increased protein levels of ER stress markers AFT4 and CHOP, and autophagy substrate p62. Oleate upregulated adipose differentiation-related protein (ADRP, a lipid droplet-associated protein), and promoted lipid droplet formation in palmitate-treated Caki-1 cells. These results indicate that Caki-1 3D spheroids are a simple and reproducible in vitro system for lipotoxicity study. It can potentially be used for evaluating human renal proximal tubular toxicity and drug screening. NIH/NIGMS R16GM149499, NIH TL1DK136047. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Persianolide-A, an eudesmanolide-type sesquiterpene lactone from Artemisia kopetdaghensis, induces apoptosis by regulating ERK signaling pathways

Background and purpose: Herbal components, particularly sesquiterpenes, are progressively recognized as a crucial resource for developing effective therapeutic agents for breast cancer. In this study, the effect of a sesquiterpene lactone known as 8-O-dihydroxy-11a,13-dihydroeudesma-4(15)-en-12,6a-olide (persianolide- A) was examined in breast cancer cell lines. Experimental approach: MDA-MB-231 and MCF-7 cancer cells were grown in DMEM solution with 10% FBS. Then, an MTT assay was performed to evaluate cell viability. Apoptosis was detected by annexin-PI staining. A caspase 3/7 activity assay kit was used to assess the activity of caspase-3 and caspase-7. Protein expression of Bcl-2, Bax, and p-ERK1/2 was determined by western blotting. Findings/Results: This study showed that the IC50 values of the persianolide-A for MCF-7 and MDA-MB- 468 cells are 34.76 and 54.48 μM, respectively. In addition, persianolide-A showed a significant increase in apoptosis in both MDAMB-231 and MCF-7 breast cancer cell lines. Persianolide-A significantly increased the expression of the pro-apoptotic protein Bax and decreased the expression of the anti-apoptotic protein Bcl-2. Also, presinolide-A treatment led to a substantial increase in caspase activity with a ratio of 3/7 in both MCF- 7 and MDA-MB-231 cancer cells. In addition, the study showed that persianolide-A decreased the expression of p-ERK1/2 protein. Conclusion and implications: The results of this study suggest that persianolide-A, sourced from Artemisia kopetdaghensis, induces cell apoptosis in breast cancer cell types. The molecular mechanisms could be implicated in the modulation of the ERK1/2 signaling pathway.

A Novel Mechanism Underlying Apoptosis Resistance in Pulmonary Arterial Smooth Cuscle Cells (PASMCs)

RATIONALE: Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling, with PASMC hyperplasia and formation of vaso-occlusive lesions. New data show PASMCs isolated from robust animal models of PH are resistant to apoptosis, a feature which may propagate disease as abnormal PASMCs evade death. Apoptosis is a highly regulated pathway resulting in caspase-3 (casp3) activation and translocation to the nucleus, where cleavage of proteins results in cell death. In several cancers the membrane protein aquaporin 1 (AQP1) plays a role in apoptosis resistance, although the mechanism remains unknown. We showed AQP1 is expressed in PASMCs and increased in rat models of PH. Therefore, we hypothesized increased AQP1 contributes to apoptosis resistance in PASMCs. METHODS: The Sugen-hypoxia (SuHx) rat model of PH was used. Rats were injected with vascular endothelial growth factor receptor type 2 inhibitor Sugen-5416 and placed in 10% O2 for 3 wk, then returned to 21% O2 for 2 wk. Control animals received vehicle injection and were kept in 21% O2. Distal PASMCs were isolated via enzymatic digestion. AQP1 was depleted in SuHx PASMCs with small interfering RNA (siAQP1), with non-targeting siRNA as control. AQP1 was overexpressed in control PASMCs with adenoviral constructs containing wild type AQP1 (AdAQP1) or green fluorescent protein (control). Cells were treated with PBS or pro-apoptotic hydrogen peroxide (500 μM; 24 h). Apoptosis was measured by Hoechst staining. Caspase activity was measured via luminescent caspase-3/7 activity assay (Caspase-Glo® 3/7) in whole cell lysates and nuclear fractions. RESULTS: Depleting AQP1 via siAQP1 restored apoptosis susceptibility in SuHx PASMCs, and overexpressing AQP1 via AdAQP1 reduced susceptibility to apoptosis in control PASMCs. At baseline, there was no difference in casp3 activity between SuHx and control total cell lysates. However, casp3 activity was lower in SuHx nuclear fractions. Under stimulated conditions, SuHx PASMCs exhibited significantly less total and nuclear casp3 activity, with nuclear activity similar to levels observed in unstimulated controls. To explore an underlying mechanism for these findings, in silico analysis of AQP1 was performed revealing 3 potential casp3 cleavage sites, suggesting the possibility these proteins could interact. To investigate a potential protein-protein interaction, proximity-based biotinylation assays were performed in PASMCs using AdAQP1 (control) or AQP1 fused to biotin ligase. Cells were incubated with cell-permeable biotin (50 μM), lysed, and biotinylated proteins precipitated and immunoblotted for casp3 and other cytosolic proteins not expected to interact with AQP1. To confirm bidirectionality, similar biotinylation assays were performed using adenoviral constructs with wild-type (control) or biotin ligase-fused casp3 and immunoblotting for AQP1. Results showed a specific AQP1/casp3 interaction in PASMCs. Furthermore, the predominant form of casp3 interacting with AQP1 appears to be inactive (37kD). CONCLUSION: We speculate that increased AQP1 in SuHx PASMCs interacts with casp3 preventing its activation and/or nuclear translocation, conferring apoptosis resistance. Future studies further characterizing the interaction between AQP1 and casp3 could provide the basis for novel therapeutic targets focused on restoring PASMC apoptosis susceptibility. F32 HL165766-01, T32 HL007534, Bauernschmidt Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Transient caspase-mediated activation of caspase-activated DNase causes DNA damage required for phagocytic macrophage differentiation

Phagocytic macrophages are crucial for innate immunity and tissue homeostasis. Most tissue-resident macrophages develop from embryonic precursors that populate every organ before birth to lifelong self-renew. However, the mechanisms for versatile macrophage differentiation remain unknown. Here, we use invivo genetic and cell biological analysis of the Drosophila larval hematopoietic organ, the lymph gland that produces macrophages. We show that the developmentally regulated transient activation of caspase-activated DNase (CAD)-mediated DNA strand breaks in intermediate progenitors is essential for macrophage differentiation. Insulin receptor-mediated PI3K/Akt signaling regulates the apoptosis signal-regulating kinase 1 (Ask1)/c-Jun kinase (JNK) axis to control sublethal levels of caspase activation, causing DNA strand breaks during macrophage development. Furthermore, caspase activity is also required for embryonic-origin macrophage development and efficient phagocytosis. Our study provides insights into developmental signaling and CAD-mediated DNA strand breaks associated with multifunctional and heterogeneous macrophage differentiation.

Macromolecules with predominant β-pleated sheet proteins in extracellular vesicles released from Raphanus sativus L. var. caudatus Alef microgreens induce DNA damage-mediated apoptosis in HCT116 colon cancer cells

Plant-derived bioactive macromolecules (i.e., proteins, lipids, and nucleic acids) were prepared as extracellular vesicles (EVs). Plant-derived EVs are gaining pharmaceutical research interest because of their bioactive components and delivery properties. The spherical nanosized EVs derived from Raphanus sativus L. var. caudatus Alef microgreens previously showed antiproliferative activity in HCT116 colon cancer cells from macromolecular compositions (predominantly proteins). To understand the mechanism of action, the biological activity studies, i.e., antiproliferation, cellular biochemical changes, DNA conformational changes, DNA damage, apoptotic nuclear morphological changes, apoptosis induction, and apoptotic pathways, were determined by neutral red uptake assay, synchrotron radiation-based Fourier transform infrared microspectroscopy, circular dichroism spectroscopy, comet assay, 4′,6-diamidino-2-phenylindole (DAPI) staining, flow cytometry, and caspase activity assay, respectively. EVs inhibited HCT116 cell growth in concentration- and time-dependent manners, with a half-maximal inhibitory concentration of 675.4 ± 33.8 μg/ml at 48 h and a selectivity index of 1.5 ± 0.076. HCT116 treated with EVs mainly changed the cellular biochemical compositions in the nucleic acids and carbohydrates region. The DNA damage caused no changes in DNA conformation. The apoptotic nuclear morphological changes were associated with the increased apoptotic cell population. The apoptotic cell death was induced by both extrinsic and intrinsic pathways. EVs have potential as antiproliferative bioparticles.

Cystatin C: immunoregulation role in macrophages infected with Porphyromonas gingivalis.

Periodontitis is a chronic infectious disease, characterized by an exacerbated inflammatory response and a progressive loss of the supporting tissues of the teeth. Porphyromonas gingivalis is a key etiologic agent in periodontitis. Cystatin C is an antimicrobial salivary peptide that inhibits the growth of P. gingivalis. This study aimed to evaluate the antimicrobial activity of this peptide and its effect on cytokine production, nitric oxide (NO) release, reactive oxygen species (ROS) production, and programmed cell death in human macrophages infected with P. gingivalis. Monocyte-derived macrophages generated from peripheral blood were infected with P. gingivalis (MOI 1:10) and stimulated with cystatin C (2.75 µg/ml) for 24 h. The intracellular localization of P. gingivalis and cystatin C was determined by immunofluorescence and transmission electron microscopy (TEM). The intracellular antimicrobial activity of cystatin C in macrophages was assessed by counting Colony Forming Units (CFU). ELISA assay was performed to assess inflammatory (TNFα, IL-1β) and anti-inflammatory (IL-10) cytokines. The production of nitrites and ROS was analyzed by Griess reaction and incubation with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), respectively. Programmed cell death was assessed with the TUNEL assay, Annexin-V, and caspase activity was also determined. Our results showed that cystatin C inhibits the extracellular growth of P. gingivalis. In addition, this peptide is internalized in the infected macrophage, decreases the intracellular bacterial load, and reduces the production of inflammatory cytokines and NO. Interestingly, peptide treatment increased ROS production and substantially decreased bacterial-induced macrophage apoptosis. Cystatin C has antimicrobial and immuno-regulatory activity in macrophages infected with P. gingivalis. These findings highlight the importance of understanding the properties of cystatin C for its possible therapeutic use against oral infections such as periodontitis.

Effects of feeding chicken egg yolk antibodies on intestinal cell apoptosis, oxidative stress and microbial flora of tilapia (Oreochromis niloticus) infected with Streptococcus agalactiae

Streptococcosis, the most common bacterial disease of fish in recent years, is highly infectious and lethal, and has become an important factor hindering the healthy and sustainable development of aquaculture. Chicken egg yolk antibody (IgY) has the advantages of high antigen specificity, inexpensive and easy to obtain, simple preparation, no toxic side effects, and in line with animal welfare, which is a green and safe alternative to antibiotics. In this study, the potential of specific IgY in the treatment of gastrointestinal pathogens was explored by observing the effects of specific IgY on intestinal flora, pathological tissue, apoptosis, oxidative stress, and inflammatory response of tilapia. We used the specific IgY prepared in the early stage to feed tilapia for 10 days, and then the tilapia was challenged with Streptococcus agalactiae. The results showed that feeding IgY before challenge had a small effect on the intestinal flora, and after challenge specific IgY decreased the proportion of Streptococcus and increased the diversity of the intestinal flora; in histopathology, specific IgY decreased tissue damage and maintained the integrity of tissue structure.Further study found that specific IgY can reduce intestinal epithelial cell apoptosis and reduce caspase activity; at the same time, the content of MDA was decreased, and the activities of SOD, CAT, GSH-Px and GR were increased. In addition, specific IgY can down-regulate the expression levels of IL-8 and TNF-α genes and up-regulate the expression levels of IL-10 and TGF-β. The results of this study showed that specific IgY could improve the intestinal flora of tilapia infected with Streptococcus agalactiae, reduce intestinal cell apoptosis, oxidative stress injury and inflammatory response, thereby reducing tissue damage and protecting the health of tilapia. Overall, specific IgY can be further explored as a potential antibiotic alternative for gastrointestinal pathogen infections.

The Role of SARS-CoV-2 Spike Protein in the Growth of Cervical Cancer Cells.

Recently developed vaccines for the SARS-CoV-2 virus utilize endogenous production of the virus' spike protein (SP), allowing the host to develop an immune response. As a result of the novelty of this virus and its vaccines, little is known overall about the potential effects of the SP on the pathogenesis of neoplasia, either from vaccination or from infection. This study was designed to investigate whether SARS-CoV-2 SP has any direct effect on SiHa cervical cancer cells. The effects of SARS-CoV-2 SP on cervical cancer cell proliferation and apoptosis were investigated by using clonogenic cell survival assay, quick cell proliferation assay, and caspase-3 activity kits in a widely-used cervical cancer cell line, SiHa. RT-PCR and immunohistochemistry were also performed to determine the potential molecular mechanisms. The growth and proliferation of SiHa cancer cells were inhibited by SARS-CoV-2 SP. SARS-CoV-2 SP also induced apoptosis in SiHa cancer cells. The anti-proliferative effect of SARS-CoV-2 SP on SiHa cancer cells was associated with the up-regulation of the anti-proliferative molecule p53. The pro-apoptotic effect of SARS-CoV-2 SP on SiHa cells was associated with the up-regulation of the pro-apoptotic molecule TRAIL. SARS-CoV-2 SP inhibits the growth of cervical cancer via up-regulation of p53 and TRAIL. Further studies are needed to elaborate on the potential effects of the SARS-CoV-2 SP on other cancer cell lines and normal physiological cell lines for comparison.

Arrhythmia onsets triggered by acute myocardial ischemia are not mediated by lysophosphoglycerides accumulation in ventricular myocardium

Lysophosphoglycerides (LPLs) have been reported to accumulate in myocardium and serve as a cause of arrhythmias in acute myocardial ischemia. However, in this study we found that LPLs level in the ventricular myocardium was decreased by the onset of acute myocardial ischemia in vivo in rats. Decreasing of LPLs level in left ventricular myocardium, but not right, was observed within 26 min of left myocardial ischemia, regardless of whether arrhythmias were triggered. Lower LPLs level in the ventricular myocardium was also observed in aconitine-simulated ventricular fibrillation (P < 0.0001) and ouabain-simulated III° atrioventricular block (P < 0.0001). Shot-lasting electric shock, e.g., ≤ 40 s, decreased LPLs level, while long-lasting, e.g., 5 min, increased it (fold change = 2.27, P = 0.0008). LPLs accumulation was observed in long-lasting myocardial ischemia, e.g., 4 h (fold change = 1.20, P = 0.0012), when caspase3 activity was elevated (P = 0.0012), indicating increased cell death, but not coincided with higher frequent arrhythmias. In postmortem human ventricular myocardium, differences of LPLs level in left ventricular myocardium was not observed among coronary artery disease- and other heart diseases-caused sudden death and non-heart disease caused death. LPLs level manifested a remarkable increasing from postmortem 12 h on in rats, thus abolishing the potential for serving as biomarkers of sudden cardiac death. Token together, in this study we found that LPLs in ventricular myocardium were initially decreased by the onset of ischemia, LPLs accumulation do not confer arrhythmogenesis during acute myocardial ischemia. It is necessary to reassess the roles of LPLs in myocardial infarction.

Impact of Prospero Homeobox-1 (PROX-1) οn the Oncogenic Phenotypes of Hepatocellular Carcinoma Cells.

Transcriptional factor prospero homeobox-1 (PROX-1) is crucial for the embryonic development of various organs and cell fate specification. It exhibits either an oncogenic or tumor suppressive activity depending on cancer types. However, the relationship between PROX-1 and hepatocellular carcinoma (HCC) remains obscure. This study was conducted to investigate the effect of PROX-1 on the invasive and oncogenic phenotypes of human HCC cells. The effect of PROX-1 on tumor cell behavior was investigated by using a pcDNA-myc vector and a small interfering RNA in HepG2 and Huh7 human HCC cell lines. Flow cytometry, migration, invasion, proliferation, and tube formation assays were performed. PROX-1 expression in human HCC cells was explored by western blotting. PROX-1 overexpression enhanced tumor cell proliferation and inhibited apoptosis and cell cycle arrest by modulating the activities of caspase-3, PARP, and cyclin-dependent kinase inhibitors, including p21, p27, and p57 in HCC cells. After PROX-1 overexpression, the number of migrating and invading HCC cells significantly increased, and the expression levels of N-cadherin and Snail increased in HCC cells. PROX-1 overexpression enhanced angiogenesis through increased VEGF-A and VEGF-C expression and decreased angiostatin expression. PROX-1 overexpression also increased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and forkhead box O1 (FOXO1) in HCC cells. After PROX-1 knockdown, their phosphorylation was reversed. PROX-1 overexpression is associated with the invasive and oncogenic phenotypes of human HCC cells via GSK-3β and FOXO1 phosphorylation.

Loureirin B improves H/R-induced hepatic ischemia-reperfusion injury by downregulating ALOX5 to regulate mitochondrial homeostasis.

This study was conceived to explore the role and the mechanism of Loureirin B (LB) in hepatic IRI. The viability of LB-treated AML-12 cells was assessed using CCK-8 assay and inflammatory cytokines were detected using ELISA. The activities of ROS and oxidative stress markers MDA, SOD, and GSH-Px were detected using DCFH-DA and corresponding assay kits. The cell apoptosis and caspase3 activity were estimated with flow cytometry and caspase3 assay kits. The expressions of arachidonate 5-lipoxygenase (ALOX5) and apoptosis- and mitochondrial dynamics-related proteins were detected using western blot. The interaction between LB and ALOX5 was analyzed with molecular docking. The transfection efficacy of oe-ALOX5 was examined with RT-qPCR and western blot. Mitochondrial membrane potential was detected with JC-1 staining and immunofluorescence (IF) assay was employed to estimate mitochondrial fusion and fission. The present work found that LB revived the viability, inhibited inflammatory response, suppressed oxidative stress, repressed the apoptosis, and maintained mitochondrial homeostasis in H/R-induced AML-12 cells, which were all reversed by ALOX5 overexpression. Collectively,LB regulated mitochondrial homeostasis by downregulating ALOX5, thereby improving hepatic IRI.

Bioactive compounds and bioactivity of the heads of wild artichokes

Wild artichokes (Cyanara algarbiensis, C. baetica, C. cardunculus, and C. humilis) heads are globally consumed, although some taxa remain understudied, constituting natural reservoirs of bioactive phytochemicals. This study was focused on four Cyanara taxa collected in diverse locations in the SE of the Iberian Peninsula in comparison with cultivated C. scolymus. Analyses of vitamin C was effected by HPLC-DAD, and the methanol:water (80:20, v/v) extracts were analyzed for: i) phenolic compounds by HPLC-DAD and LC-MS; ii) phenolic acids and flavonoid using the Folin-Ciocalteu and aluminum chloride methods; iii) the antioxidant activity through the DPPH and ABTS•+ methods; iv) the in vitro antiproliferative activity against HT-29 cells using the MTT assay; and v) caspase-3 activity, which was performed to verify induced apoptosis. Vitamin C highlighted in C. humilis (59.9 mg/100 g fw), and total phenolics (by chromatography quantitation) reached 645.9 mg/100 g fw in C. baetica. The antioxidant activity stood out in C. baetica, with 0.50 (DPPH) and 0.52 (ABTS•+) mmol TE/100 g fw. Chlorogenic acid, apigenin-7-O-glucoronide, and apigenin-7-O-rutinoside highlighted in C. baetica (159.5, 274.5, and 50.3 mg/100 g fw); and quercetin-3-O-glucoside and 1,5-di-O-caffeoylquinic acid stood out in C. algarbiensis (61.5 and 93.5 mg/100 g fw). Artichoke extracts inhibited HT-29 colorectal cancer cells proliferation, particularly the ones from C. algarbiensis and C. baetica (GI50 of 975 and 650 μg/mL for 72-h exposed cells). This work constitutes a whole approach to the knowledge of phytochemicals and bioactivities of wild artichoke taxa to discern their use as functional foods.

A novel TCGA-validated programmed cell-death-related signature of ovarian cancer

BackgroundOvarian cancer (OC) is a gynecological malignancy tumor with high recurrence and mortality rates. Programmed cell death (PCD) is an essential regulator in cancer metabolism, whose functions are still unknown in OC. Therefore, it is vital to determine the prognostic value and therapy response of PCD-related genes in OC.MethodsBy mining The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx) and Genecards databases, we constructed a prognostic PCD-related genes model and performed Kaplan-Meier (K-M) analysis and Receiver Operating Characteristic (ROC) curve for its predictive ability. A nomogram was created via Cox regression. We validated our model in train and test sets. Quantitative real-time PCR (qRT-PCR) was applied to identify the expression of our model genes. Finally, we analyzed functional analysis, immune infiltration, genomic mutation, tumor mutational burden (TMB) and drug sensitivity of patients in low- and high-risk group based on median scores.ResultsA ten-PCD-related gene signature including protein phosphatase 1 regulatory subunit 15 A (PPP1R15A), 8-oxoguanine-DNA glycosylase (OGG1), HECT and RLD domain containing E3 ubiquitin protein ligase family member 1 (HERC1), Caspase-2.(CASP2), Caspase activity and apoptosis inhibitor 1(CAAP1), RB transcriptional corepressor 1(RB1), Z-DNA binding protein 1 (ZBP1), CD3-epsilon (CD3E), Clathrin heavy chain like 1(CLTCL1), and CCAAT/enhancer-binding protein beta (CEBPB) was constructed. Risk score performed well with good area under curve (AUC) (AUC3 − year =0.728, AUC5 − year = 0.730). The nomogram based on risk score has good performance in predicting the prognosis of OC patients (AUC1 − year =0.781, AUC3 − year =0.759, AUC5 − year = 0.670). Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that the erythroblastic leukemia viral oncogene homolog (ERBB) signaling pathway and focal adhesion were enriched in the high-risk group. Meanwhile, patients with high-risk scores had worse OS. In addition, patients with low-risk scores had higher immune-infiltrating cells and enhanced expression of checkpoints, programmed cell death 1 ligand 1 (PD-L1), indoleamine 2,3-dioxygenase 1 (IDO-1) and lymphocyte activation gene-3 (LAG3), and were more sensitive to A.443,654, GDC.0449, paclitaxel, gefitinib and cisplatin. Finally, qRT-PCR confirmed RB1, CAAP1, ZBP1, CEBPB and CLTCL1 over-expressed, while PPP1R15A, OGG1, CASP2, CD3E and HERC1 under-expressed in OC cell lines.ConclusionOur model could precisely predict the prognosis, immune status and drug sensitivity of OC patients.

Neuroprotective Effect of Combined Treatment with Epigallocatechin 3-Gallate and Melatonin on Familial Alzheimer's Disease PSEN1 E280A Cerebral Spheroids Derived from Menstrual Mesenchymal Stromal Cells.

Familial Alzheimer's disease (FAD) is caused by mutations in one or more of 3 genes known as AβPP, PSEN1, and PSEN2. There are currently no effective therapies for FAD. Hence, novel therapeutics are needed. To analyze the effect of treatment with a combination of epigallocatechin-3-gallate (EGCG) and Melatonin (N-acetyl-5-methoxytryptamine, aMT) in a cerebral spheroid (CS) 3D in vitro model of PSEN 1 E280A FAD. We developed a CS in vitro model based on menstrual stromal cells derived from wild-type (WT) and mutant PSEN1 E280A menstrual blood cultured in Fast-N-Spheres V2 medium. Beta-tubulin III, choline acetyltransferase, and GFAP in both WT and mutant CSs spontaneously expressed neuronal and astroglia markers when grown in Fast-N-Spheres V2 medium for 4 or 11 days. Mutant PSEN1 CSs had significantly increased levels of intracellular AβPP fragment peptides and concomitant appearance of oxidized DJ-1 as early as 4 days, and phosphorylated tau, decreased ΔΨm, and increased caspase-3 activity were observed on Day 11. Moreover, mutant CSs were unresponsive to acetylcholine. Treatment with a combination of EGCG and aMT decreased the levels of all typical pathological markers of FAD more efficiently than did EGCG or aMT alone, but aMT failed to restore Ca2+ influx in mutant CSs and decreased the beneficial effect of EGCG on Ca2+ influx in mutant CSs. Treatment with a combination of EGCG and aMT can be of high therapeutic value due to the high antioxidant capacity and anti-amyloidogenic effect of both compounds.

Neuroprotective Effect of Sterculia setigera Leaves Hydroethanolic Extract.

Plants are a valuable source of information for pharmacological research and new drug discovery. The present study aimed to evaluate the neuroprotective potential of the leaves of the medicinal plant Sterculia setigera. In vitro, the effect of Sterculia setigera leaves dry hydroethanolic extract (SSE) was tested on cultured cerebellar granule neurons (CGN) survival when exposed to hydrogen peroxide (H2O2) or 6-hydroxydopamine (6-OHDA), using the viability probe fluorescein diacetate (FDA), a lactate dehydrogenase (LDH) activity assay, an immunocytochemical staining against Gap 43, and the quantification of the expression of genes involved in apoptosis, necrosis, or oxidative stress. In vivo, the effect of intraperitoneal (ip) injection of SSE was assessed on the developing brain of 8-day-old Wistar rats exposed to ethanol neurotoxicity by measuring caspase-3 activity on cerebellum homogenates, the expression of some genes in tissue extracts, the thickness of cerebellar cortical layers and motor coordination. In vitro, SSE protected CGN against H2O2 and 6-OHDA-induced cell death at a dose of 10µg/mL, inhibited the expression of genes Casp3 and Bad, and upregulated the expression of Cat and Gpx7. In vivo, SSE significantly blocked the deleterious effect of ethanol by reducing the activity of caspase-3, inhibiting the expression of Bax and Tp53, preventing the reduction of the thickness of the internal granule cell layer of the cerebellar cortex, and restoring motor functions. Sterculia setigera exerts neuroactive functions as claimed by traditional medicine and should be a good candidate for the development of a neuroprotective treatment against neurodegenerative diseases.

Antioxidant Activity and Mechanism of Action of Amwaprin: A Protein in Honeybee (Apis mellifera) Venom.

Bee venom contains several bioactive components, including enzymatic and non-enzymatic proteins. There is increasing interest in the bioactive components of bee venom since they have exhibited various pharmacological effects. Recently, Apis mellifera waprin (Amwaprin) was identified as a novel protein in Apis mellifera (honeybee) venom and characterized as an antimicrobial agent. Herein, the novel biological function of Amwaprin as an antioxidant is described. In addition, the antioxidant effects of Amwaprin in mammalian cells were investigated. Amwaprin inhibited the growth of, oxidative stress-induced cytotoxicity, and inflammatory response in mammalian NIH-3T3 cells. Amwaprin decreased caspase-3 activity during oxidative stress and exhibited protective activity against oxidative stress-induced cell apoptosis in NIH-3T3 and insect Sf9 cells. The mechanism underlying the cell protective effect of Amwaprin against oxidative stress is due to its direct binding to the cell membrane. Furthermore, Amwaprin demonstrated radical-scavenging activity and protected against oxidative DNA damage. These results suggest that the antioxidant capacity of Amwaprin is attributed to the synergistic effects of its radical-scavenging action and cell shielding, indicating its novel role as an antioxidant agent.