Cell Cleavage Research Articles

γ-Secretase Cleaves Bifunctional Fatty Acid-Conjugated Small Molecules with Amide Bonds in Mammalian Cells.

Connecting two small molecules, such as ligands, fluorophores, or lipids, together via a linker with amide bonds is a widely used strategy to generate synthetic bifunctional molecules for various biological and biomedical applications. Such bifunctional molecules have been used in live-cell experiments under the assumption that they should be stable in cells. However, we recently found that a membrane-targeting bifunctional molecule, composed of a lipopeptide and the small-molecule ligand trimethoprim, referred to as mgcTMP, underwent amide-bond cleavage in mammalian cells. In this work, we first identified γ-secretase as the major protease degrading mgcTMP in cells. We next investigated the intracellular degradation of several different types of amide-linked bifunctional compounds and found that N-terminally fatty acid-conjugated small molecules are susceptible to γ-secretase-mediated amide-bond cleavage. In contrast, amide-linked bifunctional molecules composed of two small molecules, such as ligands and hydrophobic groups, which lack lipid modification, did not undergo intracellular degradation. These findings highlight a previously overlooked consideration for the development and application of lipid-based bifunctional molecules in chemical biology research.

Non-canonical spatial organization of heterochromatin in mouse preimplantation embryos.

Spatial genome organization refers to the conformation of the chromosomes and their relative positioning within the nucleus. In mammals, fertilization entails intense chromatin remodeling of parental genomes, as well as large-scale structural changes in nuclear organization of the newly formed zygote over the first two cell cleavages. The molecular characteristics, mechanisms and functionality of spatial genome organization during the early steps of development in mouse have been extensively studied and will be presented in this review, with a specific focus on heterochromatin. Concomitantly to maturation of genomic architecture, the embryonic genome activation occurs in transient waves of transcription. Here, we will also discuss the putative link between heterochromatin organization and the regulation of genome expression.

Glycocalyx Interactions Modulate the Cellular Uptake of Albumin-Coated Nanoparticles.

Albumin-based nanoparticles (ABNPs) represent promising drug carriers in nanomedicine due to their versatility and biocompatibility, but optimizing their effectiveness in drug delivery requires understanding their interactions with and uptake by cells. Notably, albumin interacts with the cellular glycocalyx, a phenomenon particularly studied in endothelial cells. This observation suggests that the glycocalyx could modulate ABNP uptake and therapeutic efficacy, although this possibility remains unrecognized. In this study, we elucidate the critical role of the glycocalyx in the cellular uptake of a model ABNP system consisting of silica nanoparticles (NPs) coated with native, cationic, and anionic albumin variants (BSA, BSA+, and BSA-). Using various methodologies-including fluorescence anisotropy, dynamic light scattering, microscale thermophoresis, surface plasmon resonance spectroscopy, and computer simulations─we found that both BSA and BSA+, but not BSA-, interact with heparin, a model glycosaminoglycan (GAG). To explore the influence of albumin-GAG interactions on NP uptake, we performed comparative uptake studies in wild-type and GAG-mutated Chinese hamster ovary cells (CHO), along with complementary approaches such as enzymatic GAG cleavage in wild-type cells, chemical inhibition, and competition assays with exogenous heparin. We found that the glycocalyx enhances the cell uptake of NPs coated with BSA and BSA+, while serving as a barrier to the uptake of NPs coated with BSA-. Furthermore, we showed that harnessing albumin-GAG interactions increases cancer cell death induced by paclitaxel-loaded albumin-coated NPs. These findings underscore the importance of albumin-glycocalyx interactions in the rational design and optimization of albumin-based drug delivery systems.

Stepwise release of Activin-A from its inhibitory prodomain is modulated by cysteines and requires furin coexpression to promote melanoma growth

The Activin-A precursor dimer can be cleaved by furin, but how this proteolytic maturation is regulated in vivo and how it facilitates access to signaling receptors is unclear. Here, analysis in a syngeneic melanoma grafting model shows that without furin coexpression, Activin-A failed to accelerate tumor growth, correlating with failure of one or both subunits to undergo cleavage in signal-sending cells, even though compensatory processing by host cells nonetheless sustained elevated circulating Activin-A levels. In reporter assays, furin-independent cleavage of one subunit enabled juxtacrine Activin-A signaling, whereas completion of proteolytic maturation by coexpressed furin or by recipient cells stimulated contact-independent activity, crosstalk with BMP receptors, and signal inhibition by follistatin. Mechanistically, Activin-A processing was modulated by allosteric disulfide bonds flanking the furin site. Disruption of these disulfide linkages with the prodomain enabled Activin-A binding to cognate type II receptors independently of proteolytic maturation. Stepwise proteolytic maturation is a novel mechanism to control Activin-A protein interactions and signaling.

Advances in the cell biology of the trafficking and processing of amyloid precursor protein: impact of familial Alzheimer's disease mutations.

The production of neurotoxic amyloid-β peptides (Aβ) is central to the initiation and progression of Alzheimer's disease (AD) and involves sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. APP and the secretases are transmembrane proteins and their co-localisation in the same membrane-bound sub-compartment is necessary for APP cleavage. The intracellular trafficking of APP and the β-secretase, BACE1, is critical in regulating APP processing and Aβ production and has been studied in several cellular systems. Here, we summarise the intracellular distribution and transport of APP and its secretases, and the intracellular location for APP cleavage in non-polarised cells and neuronal models. In addition, we review recent advances on the potential impact of familial AD mutations on APP trafficking and processing. This is critical information in understanding the molecular mechanisms of AD progression and in supporting the development of novel strategies for clinical treatment.

EP4 receptor agonist CAY10598 upregulates ROS-dependent Hsp90 cleavage in colorectal cancer cells

Prostaglandin E2 (PGE2) interacts with four specific G protein-coupled receptors, namely EP1, EP2, EP3, and EP4, playing a pivotal role in determining the fate of cells. Our previous findings highlighted that stimulating the EP4 receptor with its agonist, CAY10598, triggers apoptosis in colon cancer HCT116 cells via the production of reactive oxygen species (ROS). This process also reduces the phosphorylation of the oncogenic protein JAK2 and leads to its degradation in these cells. In this study, our goal was to explore the pathways through which CAY10598 leads to JAK2 degradation. We focused on Hsp90, a heat shock protein family member known for its role as a molecular chaperone maintaining the stability of several key proteins including EGFR, MET, Akt, and JAK2. Our results show that CAY10598 decreases the levels of client proteins of Hsp90 in HCT116 cells, an effect reversible by pretreatment with the ROS scavenger N-acetyl cysteine (NAC) or the proteasome inhibitor MG132, indicating that the degradation is likely driven by ROS. Furthermore, we observed that CAY10598 cleaves both α and β isoforms of Hsp90, the process inhibited by NAC. Inhibition of EP4 with the antagonist GW627368x not only prevented the degradation of Hsp90 client proteins but also the cleavage of Hsp90 itself in CAY10598-treated HCT116 cells. Additionally, CAY10598 suppressed the growth of HCT116 cells implanted in mice. Our findings reveal that CAY10598 induces apoptosis in cancer cells by a novel mechanism involving the ROS-dependent cleavage of Hsp90, thereby inhibiting the function of crucial Hsp90 client proteins.

Human NQO1 as a Selective Target for Anticancer Therapeutics and Tumor Imaging.

Human NAD(P)H-quinone oxidoreductase1 (HNQO1) is a two-electron reductase antioxidant enzyme whose expression is driven by the NRF2 transcription factor highly active in the prooxidant milieu found in human malignancies. The resulting abundance of NQO1 expression (up to 200-fold) in cancers and a barely detectable expression in body tissues makes it a selective marker of neoplasms. NQO1 can catalyze the repeated futile redox cycling of certain natural and synthetic quinones to their hydroxyquinones, consuming NADPH and generating rapid bursts of cytotoxic reactive oxygen species (ROS) and H2O2. A greater level of this quinone bioactivation due to elevated NQO1 content has been recognized as a tumor-specific therapeutic strategy, which, however, has not been clinically exploited. We review here the natural and new quinones activated by NQO1, the catalytic inhibitors, and the ensuing cell death mechanisms. Further, the cancer-selective expression of NQO1 has opened excellent opportunities for distinguishing cancer cells/tissues from their normal counterparts. Given this diagnostic, prognostic, and therapeutic importance, we and others have engineered a large number of specific NQO1 turn-on small molecule probes that remain latent but release intense fluorescence groups at near-infrared and other wavelengths, following enzymatic cleavage in cancer cells and tumor masses. This sensitive visualization/quantitation and powerful imaging technology based on NQO1 expression offers promise for guided cancer surgery, and the reagents suggest a theranostic potential for NQO1-targeted chemotherapy.

Naringin Induces ROS-Stimulated G1 Cell-Cycle Arrest and Apoptosis in Nasopharyngeal Carcinoma Cells.

Naringin, a bioflavonoid compound from grapefruit or citrus, exerts anticancer activities on cervical, thyroid, colon, brain, liver, lung, thyroid, and breast cancers. The present investigation addressed exploring the anticancer effects of naringin on nasopharyngeal carcinoma (NPC) cells. Naringin exhibits a cytotoxic effect on NPC-TW 039 and NPC-TW 076 cells with IC50 372/328 and 394/307 μM for 24 or 48 h, respectively, while causing little toxicity toward normal gingival epithelial (SG) cells (>500/500 μM). We established that naringin triggered G1 arrest is achieved by suppressing cyclin D1, cyclin A, and CDK2, and upregulating p21 protein in NPC cells. Exposure of NPC cells to naringin caused a series of events leading to apoptosis including morphology change (cell shrinkage and membrane blebbing) and chromatin condensation. Annexin V and PI staining indicated that naringin treatment promotes necrosis and late apoptosis in NPC cells. DiOC6 staining showed a decline in the mitochondrial membrane potential by naringin treatment, which was followed with cytochrome c release, Apaf-1/caspase-9/-3 activation, PARP cleavage, and EndoG expression in NPC cells. Naringin upregulated proapoptotic Bax and decreased antiapoptotic Bcl-xL expression, and dysregulated Bax/Bcl-xL ratio in NPC cells. Notably, naringin enhanced death receptor-related t-Bid expression. Furthermore, an increased Ca2+ release by naringin treatment which instigated endoplasmic reticulum stress-associated apoptosis through increased IRE1, ATF-6, GRP78, GADD153, and caspase-12 expression in NPC cells. In addition, naringin triggers ROS production, and inhibition of naringin-induced ROS generation by antioxidant N-acetylcysteine resulted in the prevention of G1 arrest and apoptosis in NPC cells. Naringin-induced ROS-mediated G1 arrest and mitochondrial-, death receptor-, and endoplasmic reticulum stress-mediated apoptosis may be a promising strategy for treating NPC.

O-248 Novel early cleavage parameters improve embryo selection and reach ploidy predictive values comparable to artificial intelligence

Abstract Study question Can we improve embryo selection and ploidy prediction using novel morphokinetic parameters in blastocyst development, and obtain results comparable to artificial intelligence? Summary answer Time and pattern of novel parameter st2 are related to ploidy. Including them in embryo evaluation improves ploidy prediction, reaching predictive values comparable to AI. What is known already Time-lapse has allowed us to detect more morphokinetic parameters and improve embryo grading systems. However, ploidy prediction still relies on invasive techniques to obtain a reliable result. Artificial intelligence is promising and can already predict blastulation or implantation with good results, but not ploidy yet. Moreover, AI presents some other limitations now, including the black box behind its decisions, which doesn’t allow clinicians to know what parameters are being considered the most. Improving the manual evaluation tools used by embryologists can help improve clinical outcomes until AI is fully introduced in IVF. Study design, size, duration A single center retrospective study was performed. Data included a total of 608 blastocyst from 106 treatments between January 2018 and July 2022. Participants/materials, setting, methods 160 treatments of patients for preimplantation genetic testing cycles were studied. Embryos were cultured in time-lapse incubators and were studied for both morphologic and kinetic parameters. 608 of the embryos developed to good quality blastocyst and were able to be biopsied, and therefore analyzed by PGT for aneuploidies on Day5 o Day6. Annotation of several morphokinetic characteristics was performed on all analyzed embryos. The study was blinded for ploidy and all clinical outcomes. Main results and the role of chance Improved embryo grading systems used by embryologist can reach high predictive values comparable to artificial intelligence. We present a new parameter, “st2, start of t2”, which refers to the first cytoplasmic movements detected at the beginning of the first cell cleavage, as highly implicated in ploidy status. Earlier st2 is associated to better euploidy rates (p < 0.001). Not only the time, but also the phenotype of the movements appears as related to ploidy. Embryos showing none or un-patterned random cytoplasmic movements show a marked tendency towards aneuploidy. Contrary, circular waves prior to cell division are highly associated with euploidy (90% of the cases) (p < 0.0001). The implication of the described parameters was also confirmed by logistic regression, with a receiver operating curve (ROC) value of 0.69 for ploidy prediction (95% confidence interval (CI), 0.62 to 0.76). We processed raw time-lapse images with an automatic annotation software using artificial intelligence and obtained a ploidy predictive value of 0.64. This means that using the optimal parameters in manual embryo evaluation, we are able to improve our grading systems and reach ploidy predictive values comparable than with AI. Limitations, reasons for caution This is a retrospective study. The ploidy predictive potential is now being tested on a prospective study. Wider implications of the findings Optimizing the indicators to select the most suitable blastocyst, like including st2, can help reducing the time until the pregnancy of an euploid baby, avoiding invasive and expensive methods. Moreover, by combining AI with the novel parameter st2 we could improve the potential of the ploidy predictions. Trial registration number not applicable

P-163 Does severe endometriosis (SE) have an effect on embryo morphokinetic parameters?

Abstract Study question Does severe endometriosis (SE) have an effect on embryo morphokinetic parameters? Summary answer Severe endometriosis may have an effect on embryonic cell divisions and could change morphokinetic parameters of in vitro developing embryos. What is known already Endometriosis is one of the leading causes of infertility that affects approximately one third of women who apply for in vitro fertilization (IVF) treatment. Controversial results were reported regarding the effect of endometriosis on both morphological and dynamic characteristics of embryos so far. Study design, size, duration This was a retrospective comparative analysis including a total of 1280 embryos (SE: 729, Tubal factor (control): 551) of 241 patients (SE: 134, control: 107) that were incubated at Time-Lapse Monitoring System (TLM) in Memorial Sisli Hospital, ART and Reproductive Genetics Center between October 2011 and July 2023. Patients with ≥ 38 years, BMI ≥ 30 and partners having severe male factor (sperm concentration: <5 mil/ml) were excluded from the study. Participants/materials, setting, methods Severe endometriosis was defined as Grade III and IV according to ASRM classification. IMSI (Intracytoplasmic Morphologically Selected Sperm Injection) was performed to all oocytes. Embryos were evaluated according to Gardner’s classification. Fertilization rates were assessed using Chi-square test. Morphokinetic parameters of embryos were compared by Student’s t-test using SPSS 28.0. Main results and the role of chance There was a homogenous distribution in terms of female age, BMI, basal FSH, AMH levels, total and mature (MII) oocyte count between study groups. Abnormal fertilization rate was higher in SE group than controls ( 4.3% vs 2.4%, p = 0.035). All morphokinetic parameters were found significantly delayed in the SE group than in the control group (p < 0.05), however, no statistical significance was obtained in terms of tSC and tEB (p > 0.05). Duration of first, second and third embryo cell cycles (ECC1, ECC2, and ECC3), synchronization of cell divisions (S2: t4-t3) and cleavage patterns (S3: t8-t5) were also found statistically significant between SE and control groups (p < 0.05). Limitations, reasons for caution The major limitation of this small-sized, retrospective study is that clinical outcomes of the embryos were not assessed. Wider implications of the findings Delayed development was observed in the embryos of SE patients. Prospective studies with larger cohorts are needed to have a better understanding between severe endometriosis and embryo morphokinetics. Trial registration number Not applicable

Diospyros rhodocalyx Kurz induces mitochondrial-mediated apoptosis via BAX, Bcl-2, and caspase-3 pathways in LNCaP human prostate cancer cell line.

Prostate cancer (PCa) is one of the causes of death in men worldwide. Although treatment strategies have been developed, the recurrence of the disease and consequential side effects remain an essential concern. Diospyros rhodocalyx Kurz, a traditional Thai medicine, exhibits diverse therapeutic properties, including anti-cancer activity. However, its anti-cancer activity against prostate cancer has not been thoroughly explored. This study aims to evaluate the anti-cancer activity and underlying mechanisms of the ethyl acetate extract of D. rhodocalyx Kurz (EADR) related to apoptosis induction in the LNCaP human prostate cancer cell line. Ethyl acetate was employed to extract the dried bark of D. rhodocalyx Kurz. The cytotoxicity of EADR on both LNCaP and WPMY-1 cells (normal human prostatic myofibroblast cell line) was evaluated using MTS assay. The effect of EADR on the cell cycle, apoptosis induction, and alteration in mitochondrial membrane potential (MMP) was assessed by the staining with propidium iodide (PI), Annexin V-FITC/PI, and JC-1 dye, respectively. Subsequent analysis was conducted using flow cytometry. The expression of cleaved caspase-3, BAX, and Bcl-2 was examined by Western blotting. The phytochemical profiling of the EADR was performed using gas chromatography-mass spectrometry (GC-MS). EADR exhibited a dose-dependent manner cytotoxic effect on LNCaP cells, with IC50 values of 15.43 and 12.35 µg/mL after 24 and 48 h, respectively. Although it also exhibited a cytotoxic effect on WPMY-1 cells, the effect was comparatively lower, with the IC50 values of 34.61 and 19.93 µg/mL after 24 and 48 h of exposure, respectively. Cell cycle analysis demonstrated that EADR did not induce cell cycle arrest in either LNCaP or WPMY-1 cells. However, it significantly increased the sub-G1 population in LNCaP cells, indicating a potential induction of apoptosis. The Annexin V-FITC/PI staining indicated that EADR significantly induced apoptosis in LNCaP cells. Subsequent investigation into the underlying mechanism of EADR-induced apoptosis revealed a reduction in MMP as evidenced by JC-1 staining. Moreover, Western blotting demonstrated that EADR treatment resulted in the upregulation of BAX, downregulation of BCL-2, and elevation of caspase-3 cleavage in LNCaP cells. Notably, the epilupeol was a prominent compound in EADR as identified by GC-MS. The EADR exhibits anti-cancer activity against the LNCaP human prostate cancer cell line by inducing cytotoxicity and apoptosis. Our findings suggest that EADR promotes apoptosis by upregulating pro-apoptotic BAX, whereas downregulation of anti-apoptotic Bcl-2 results in the reduction of MMP and the activation of caspase-3. Of particular interest is the presence of epilupeol, a major compound identified in EADR, which may hold promise as a candidate for the development of therapeutic agents for prostate cancer.

Single-base tiled screen unveils design principles of PspCas13b for potent and off-target-free RNA silencing

The development of precise RNA-editing tools is essential for the advancement of RNA therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) PspCas13b is a programmable RNA nuclease predicted to offer superior specificity because of its 30-nucleotide spacer sequence. However, its design principles and its on-target, off-target and collateral activities remain poorly characterized. Here, we present single-base tiled screening and computational analyses that identify key design principles for potent and highly selective RNA recognition and cleavage in human cells. We show that the de novo design of spacers containing guanosine bases at precise positions can greatly enhance the catalytic activity of inefficient CRISPR RNAs (crRNAs). These validated design principles (integrated into an online tool, https://cas13target.azurewebsites.net/) can predict highly effective crRNAs with ~90% accuracy. Furthermore, the comprehensive spacer–target mutagenesis revealed that PspCas13b can tolerate only up to four mismatches and requires ~26-nucleotide base pairing with the target to activate its nuclease domains, highlighting its superior specificity compared to other RNA or DNA interference tools. On the basis of this targeting resolution, we predict an extremely low probability of PspCas13b having off-target effects on other cellular transcripts. Proteomic analysis validated this prediction and showed that, unlike other Cas13 orthologs, PspCas13b exhibits potent on-target activity and lacks collateral effects.

HtrA-Dependent E-Cadherin Shedding Impairs the Epithelial Barrier Function in Primary Gastric Epithelial Cells and Gastric Organoids.

Impaired E-cadherin (Cdh1) functions are closely associated with cellular dedifferentiation, infiltrative tumor growth and metastasis, particularly in gastric cancer. The class-I carcinogen Helicobacter pylori (H. pylori) colonizes gastric epithelial cells and induces Cdh1 shedding, which is primarily mediated by the secreted bacterial protease high temperature requirement A (HtrA). In this study, we used human primary epithelial cell lines derived from gastroids and mucosoids from different healthy donors to investigate HtrA-mediated Cdh1 cleavage and the subsequent impact on bacterial pathogenesis in a non-neoplastic context. We found a severe impairment of Cdh1 functions by HtrA-induced ectodomain cleavage in 2D primary cells and mucosoids. Since mucosoids exhibit an intact apico-basal polarity, we investigated bacterial transmigration across the monolayer, which was partially depolarized by HtrA, as indicated by microscopy, the analyses of the transepithelial electrical resistance (TEER) and colony forming unit (cfu) assays. Finally, we investigated CagA injection and observed efficient CagA translocation and tyrosine phosphorylation in 2D primary cells and, to a lesser extent, similar effects in mucosoids. In summary, HtrA is a crucially important factor promoting the multistep pathogenesis of H. pylori in non-transformed primary gastric epithelial cells and organoid-based epithelial models.

Effect of cell diameter variations on the mechanical behavior of aluminum foams: Experiments and modeling

Aluminum foams can be manufactured in a variety of cell sizes; however, existing models focus primarily on density and neglect the effect of cell size. Furthermore, aluminum foams with different cell diameters exhibit diverse mechanical properties. In this study, a quasi-static/dynamic compression test was performed to investigate the mechanical behavior, damage patterns, and damage mechanisms of aluminum foams with different cell diameters. The results demonstrate that the compressive properties of aluminum foams undergo significant changes. As the cell diameter increases, normalized initial damage stress and Young's modulus also increase, leading to enhanced energy absorption. However, this increase in cell diameter negatively affects the energy absorption efficiency, resulting in increased instabilities. A finite element analysis was conducted using the LS-DYNA finite element software to investigate the behavior of aluminum foils under all the experimentally tested loading regimes. The simulation results reveal that the deformation zone formed due to collapse diminishes with increasing cell diameter. More importantly, a new damage mechanism named central cell cleavage was reported, which increased the energy absorption capacity and amplified the instability during deformation. This study offers novel insights into the mechanical behaviors exhibited by aluminum foams, thereby facilitating their application in structural engineering.

Abstract 147: Monocyte-derived Extracellular Delta-like 4 Contributes To Pulmonary Hypertension Progression By Inhibiting Endothelial Notch1 Activation

Background: We published that reduced Notch1 cleavage in lung endothelial cells (ECs) contributes to pulmonary hypertension (PH) development. However, the underlying mechanism of Notch1 inactivation remains elusive. We recently found enhanced circulating extracellular Delta-like ligand 4 (exDll4) from monocytes in HIV patients, the population with persistent systemic inflammation. Mobilized Notch ligands have demonstrated the ability to inhibit Notch signaling in both in vitro and in vivo studies. Hence, we hypothesize that inflammation increases monocyte-derived exDll4 and diminishes Notch1 cleavage in lung ECs, subsequently promoting the progression of PH. Methods and Results: Tumor necrosis factor-alpha (TNFα) is a major cytokine increase in the plasma of PAH patients and mouse models. Our in vitro studies revealed that TNFα induced a 17.1-fold and 23.6-fold increase of Dll4 mRNA and protein in human monocytes and a 7.2-fold elevation in exDll4 secretion. In PH mouse models induced by chronic hypoxia, Dll4 protein expression in circulating monocytes increased by 4.2 folds, and plasma exDll4 levels rose by 2.6 folds. In PAH patients, serum exDll4 was elevated by 3 folds compared to non-PAH patients. Mimicking enhanced exDll4 in PH patients; we found that exDll4 injection exacerbated PH progression in hypoxia-treated mice, accompanied by inhibited EC-Notch1 cleavage, leaky EC cell-cell junctions, and increased immune cell infiltration in the lungs. Notably, specific knock-out monocyte Dll4 in mice (mDll4 KO mice) significantly attenuated PH progression, indicated by reduced right ventricular systolic pressure and right ventricular remodeling. Mechanistically, exDll4 treatment significantly inhibited Notch1 activation and Notch target gene expression in human pulmonary microvascular ECs (HPMECs) and led to cell apoptosis, cell arrest, impaired barrier function, and enhanced inflammation. Conclusion: Our study demonstrates the role of monocyte-derived exDll4 in PH progression by inducing EC arrest and apoptosis and impairing barrier function in lung ECs through inhibition of Notch1 signaling. These findings imply that monocyte-derived Dll4 could be a potential therapeutic target and biomarker for PH.

Paris saponin VII induces Caspase-3/GSDME-dependent pyroptosis in pancreatic ductal adenocarcinoma cells by activating ROS/Bax signaling

ObjectiveParidis Rhizoma (Chonglou in Chinese), a traditional Chinese herbal medicine, has been shown have strong anti-tumor effects. Paris saponin VII (PSVII), an active constituent isolated from Paridis Rhizoma, was demonstrated to significantly suppress the proliferation of BxPC-3 cells in our previous study. Here, we aimed to elucidate the anti-pancreatic ductal adenocarcinoma (PDAC) effect of PSVII and the underlying mechanism. MethodsCell viability was determined by CCK-8, colony formation, and cell migration assays. Cell apoptosis and reactive oxygen species (ROS) production were measured by flow cytometry with annexin V/propidine iodide (Annexin V/PI) and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), respectively. Pyroptosis was evaluated by morphological features, Hoechst 33342/PI staining assay, and release of lactate dehydrogenase (LDH). JC-1 fluorescent dye was employed to measure mitochondrial membrane potential. Western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to determine the levels of proteins or mRNAs. The effect in vivo was assessed by a xenograft tumor model. ResultsPSVII inhibited the viability of PDAC cells (BxPC-3, PANC-1, and Capan-2 cells) and induced gasdermin E (GSDME) cleavage, as well as the simultaneous cleavage of Caspase-3 and poly (ADP-ribose) polymerase 1 (PARP). Knockdown of GSDME shifted PSVII-induced pyroptosis to apoptosis. Additionally, the effect of PSVII was significantly attenuated by Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (Z-DEVD-FMK), on the induction of GSDME-dependent pyroptosis. PSVII also elevated intracellular ROS accumulation and stimulated Bax and Caspase-3/GSDME to conduct pyroptosis in PDAC cells. The ROS scavenger N-acetyl cysteine (NAC) suppressed the release of LDH and inhibited Caspase-9, Caspase-3, and GSDME cleavage in PDAC cells, ultimately reversing PSVII-induced pyroptosis. Furthermore, in a xenograft tumor model, PSVII markedly suppressed the growth of PDAC tumors and induced pyroptosis. ConclusionThese results demonstrated that PSVII exerts therapeutic effects through Caspase-3/GSDME-dependent pyroptosis and may constitute a novel strategy for preventing chemotherapeutic resistance in patients with PDAC in the future.

Association between embryo morphokinetic development and intracytoplasmic sperm injection with epididymal sperm via time-lapse imaging.

The objective of this study was to investigate the impact of sperm source on embryo morphokinetics and the clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles by considering the clustering of data (multiple embryos per patient that share a comparable developmental timing). This matched cohort study was performed at a private university-affiliated in vitro fertilization center. Women who underwent ICSI with epididymal sperm between January 2019 and December 2020 (the percutaneous epididymal sperm aspiration group, n = 32 cycles) were matched with women who underwent ICSI with ejaculated sperm because of idiopathic male factor infertility (the male factor infertility [MFI] group, n = 32 cycles) or female infertility (the control group, n = 32 cycles). Embryos were cultured in a time-lapse imaging incubator, and morphokinetic development was recorded and compared among the groups. Significantly slower divisions were observed in embryos derived from epididymal sperm than in those derived from the MFI and control groups. Embryos derived from epididymal sperm had a significantly lower KIDScore (3.1 ± 0.2) than did those derived from ejaculated spermatozoa from the MFI (5.4 ± 0.1) and control (5.6 ± 0.2, p < 0.001) groups. Epididymal sperm-derived embryos showed a significantly greater occurrence of multinucleation (23.2%) than did those derived from ejaculated sperm from the MFI and control groups (2.8% and 3.7%, p < 0.001, respectively). Epididymal sperm-derived embryos were significantly more likely to undergo direct or reverse cleavage (11.1%) than ejaculated sperm-derived embryos in the control group (4.3%, p = 0.001). In conclusion, delayed cell cleavage and increased incidences of blastomere multinucleation and abnormal cleavage patterns are observed when epididymal-derived sperm are used for ICSI.

In Vitro Wound Healing and Anticancer Effects of Ixora coccinea in Malignant Melanoma Cell Lines.

Background Ixora coccinea is a medicinal plant with many active constituents that are responsible for wound healing and have anticancer properties. Herbal extracts increase the mechanisms related to wound healing, like blood clotting, fighting infection, and epithelialization. The effect responsible for this property may be the presence of phytoconstituents like flavonoids, polyphenols, and alkaloids. Many researchers have evaluated the wound-healing effect of I. coccinea leaf extract in aqueous methanol. This study aimed to determine the in vitro wound healing and anticancer efficacy of I. coccinea leaf ethyl acetate extract and evaluate the in silico docking of the selected phytoconstituents of I. coccinea in the 2vcj protein. Materials and methods The human dermal fibroblast cell line was used to determine the rates of cell migration and proliferation for evaluating the wound-healing effect of the I. coccinea leaf ethyl acetate fraction. 4',6-diamidino-2-phenylindole (DAPI)fluorescence labeling was used to estimate the rate of cell migration. The one-step TUNEL (TdT-mediated dUTP Nick-End Labeling) in situ apoptosis kit and the annexin V-FITC/7-AAD apoptosis kit were used to perform DNA damage assays in the malignant melanoma cell line. The ethyl acetate fraction of I. coccinea leaves was analyzed for its impact on wound healing markers, including keratin-10, keratin-14, type IV collagen, and α-SMA. Results The wound-healing nature was interesting in the ethyl acetate fraction at doses of 50 µg/mL and 100 µg/mL. Both studies involved in the DNA damage study against malignant melanoma cell lines showed the cleavage of apoptotic cancer cells, which was detected using a fluorescence microscope. When compared with the control, a dose of 100 μg/ml of ethyl acetate fraction from the leaves of I. coccinea showed fibroblast migration of cells into the wound area. The statistical values were considered significant at the level of P < 0.05. An in silico docking study on the 2vcj protein revealed that selected phytoconstituents of I. coccinea resulted in good docking scores to inhibit Hsp90. Conclusion I. coccinea ethyl acetate leaf extract can inhibit the growth of malignant melanoma cell lines and promote wound healing, as shown by the study results. It might be a viable therapeutic modality for skin cancer.

GW501516-Mediated Targeting of Tetraspanin 15 Regulates ADAM10-Dependent N-Cadherin Cleavage in Invasive Bladder Cancer Cells.

Bladder cancer aggressiveness is correlated with abnormal N-cadherin transmembrane glycoprotein expression. This protein is cleaved by the metalloprotease ADAM10 and the γ-secretase complex releasing a pro-angiogenic N-terminal fragment (NTF) and a proliferation-activating soluble C-terminal fragment (CTF2). Tetraspanin 15 (Tspan15) is identified as an ADAM10-interacting protein to induce selective N-cadherin cleavage. We first demonstrated, in invasive T24 bladder cancer cells, that N-cadherin was cleaved by ADAM10 generating NTF in the extracellular environment and leaving a membrane-anchored CTF1 fragment and that Tspan15 is required for ADAM10 to induce the selective N-cadherin cleavage. Targeting N-cadherin function in cancer is relevant to preventing tumor progression and metastases. For antitumor molecules to inhibit N-cadherin function, they should be complete and not cleaved. We first showed that the GW501516, an agonist of the nuclear receptor PPARβ/δ, decreased Tspan15 and prevented N-cadherin cleavage thus decreasing NTF. Interestingly, the drug did not modify ADAM10 expression, which was important because it could limit side effects since ADAM10 cleaves numerous substrates. By targeting Tspan15 to block ADAM10 activity on N-cadherin, GW501516 could prevent NTF pro-tumoral effects and be a promising molecule to treat bladder cancer. More interestingly, it could optimize the effects of the N-cadherin antagonists those such as ADH-1 that target the N-cadherin ectodomain.

Combined computational and synthetic strategies for the development of potent pyrazolo-pyridine derivatives as anticancer agents

PI3K comprise a group of lipid kinases mainly made up of three classes and have a particular role in signal transduction. The aberration of class IA PI3K is most frequently shown in cancer. The computation techniques have been applied for the identification of potent compounds. This involved the generation of a library of novel compounds (2700) through R-group enumeration study. The screening process utilized a combination of pharmacophore-based virtual screening and various docking algorithms, leading to the discovery of ten novel compounds. The docking studies against PI3K kinase highlighted MSA6 and MSA7 as the most promising compounds, with docking scores of -9.03 and -9.0 kcal/mol, respectively. Results of docking studies of all derivatives confirmed satisfactory binding mode of the compounds within the active site of the target protein PI3K kinase. The screened pyrazolo-pyridine derivatives (MSA1-MSA10) were synthesized and evaluated as anti-cancer agents against NSCLC using A549 cell line. MSA6 showed the dose dependent enhancement in ROS production at concentration of 10 and 20 µM in A549 cells. MSA6 triggered caspase 3 cleavage in lung cancer cells in a dose-dependent manner. MSA6 treatment at 10 and 20 µM levels boosted NF-κβ expression in the cytoplasm while decreasing it in the nucleus, as demonstrated by flow cytometry with an anti-NF-κβ antibody. The modulation of PI3K activity have been observed at 10 µM and 20 µM concentration of MSA6. A549 cells treated with MSA6 with 20 µM showed significant reduction in wound healing in cancerous cell. A549 cells treated with 20 µM showed significantly reduction in number of colonies compared to control group. Compound MSA6 having a methyl group at the para position of phenyl ring at imine carbon and phenyl ring substitution at nitrogen of pyrazole ring exhibited remarkable growth inhibitory activity with IC50 value of 49.23 μM. Based on the above results, pyrazolo-pyridine derivatives can be used for the further development of novel PI3K kinase inhibitors as anticancer agents.