Search

A central challenge in the quest for precise gene regulation within mammalian cells is the development of regulatory networks that can achieve perfect adaptation-where outputs consistently return to a set baseline post-stimulus. Here, we present such a system that leverages the CRISPR activation (CRISPRa) and anti-CRISPR proteins as two antithetic elements to establish perfect adaptation in mammalian cells and dynamically regulate gene expression. We demonstrate that this system can maintain stable expression levels of target genes in the face of external perturbations, thus providing a robust platform for biological applications. The versatility of our system is further showcased through its integration with endogenous regulatory mechanisms in T cells, such as the NF-κB-mediated immune response, and its ability to program apoptosis responses for precise spatial and temporal control of cellular growth and death. This study not only advances our understanding of gene regulation in mammalian cells but also opens new avenues for therapeutic intervention, particularly in diseases characterized by dysregulated gene expression., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Metal oxide nanoparticles(NPs) contain unique properties which have made them attractive agents in cancer treatment. The CuO nanoparticles were green synthesized using walnut shell powder in different calcination temperatures (400°, 500°, 700°, and 900 °C). The CuO nanoparticles are characterized by FTIR, XRD, BET, SEM and DLS analyses. SEM and DLS analyses showed that by increasing the required calcination temperature for synthesizing the NPs, their size was increased. DPPH analysis displayed no significant anti-oxidative properties of the CuO NPs. The MTT analysis showed that all synthesized CuO NPs exhibited cytotoxic effects on MCF-7, HCT-116, and HEK-293 cell lines. Among the CuO NPs, the CuO-900 NPs showed the least cytotoxic effect on the HEK-293 cell line (IC 50  = 330.8 µg/ml). Hoechst staining and real-time analysis suggested that the CuO-900 NPs induced apoptosis by elevation of p53 and Bax genes expression levels. Also, the CuO-900 NPs increased the Nrf-2 gene expression level in MCF-7 cells, despite the HCT-116 cells. As can be concluded from the results, the CuO-900 NPs exerted promising cytotoxic effects on breast and colon cancer cells., (© 2024. The Author(s).)

Overexpression of Bcl-2 protein is a predominant hallmark of disturbed apoptotic pathway in most of the cancers. Herein, chromone-linked thiazolidinediones were designed and synthesized to target Bcl-2 for regulating anti-apoptotic proteins. The study on in vitro cancer cell lines revealed the presence of compounds 8a, 8k, 8l, and 8n, which were found to have good to moderate anti-proliferative activity (with an IC 50 concentration less than 10 µM). Among them, 8l depicted the highest cytotoxicity on the A549 cell line with an IC 50 of 6.1 ± 0.02 µM. Aberrantly, the compounds displayed less toxicity towards human embryonic kidney HEK cells underlining its selectivity. The DCFDA study revealed a gradual increase in the ROS generation of 8l, followed by its quantification by flow analysis. Similarly, the studies including DAPI, AO/EtBr and Annexin-V binding clearly elucidated the DNA damage, membrane integrity prospects, and insights for early and late apoptotic phases. Markedly, the Bcl-2-FITC anti-body study revealed that compound 8l reduced the expression of anti-apoptotic proteins by 79.1 % compared to the control at 9 µM concentration. In addition, the molecular docking study provided the impending scope of these hybrids, showing promising interaction with the Mcl-1 target (member of the Bcl-2 family) with comparable binding affinities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Reactive oxygen species (ROS) are highly reactive and their accumulation causes oxidative damage to cells. Cells maintain survival upon mild oxidative stress with anti-oxidative systems, such as the kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system. On the other hand, upon severe oxidative stress, cells undergo regulated cell death, including apoptosis, for eliminating damaged cells. To execute efficient cell death, cells need to turn off the anti-oxidant systems, while triggering cell death. However, it remains unknown how cells orchestrate these two conflicting systems under excessive oxidative stress. Herein, we show that when cells are exposed to excessive oxidative damage, an E3 ubiquitin ligase Roquin-2 (also known as RC3H2) plays a key role in switching cell fate from survival to death by terminating activation of transforming growth factor-β-activated kinase 1 (TAK1), a positive regulator for Nrf2 activation. Roquin-2 interacted with TAK1 via four cysteine residues in TAK1 (C96, C302, C486, and C500) that are susceptible to oxidative stress and participate in oligomer formation via disulfide bonds, promoting K48-linked polyubiquitination and degradation of TAK1. Nrf2 was inactivated upon lethal oxidative stress in wild-type mouse embryonic fibroblast (MEF) cells, whereas it sustained activation and conferred resistance to Roquin-2 deficient cells, which was reversed by pharmacological or genetic inhibition of TAK1. These data demonstrate that in response to excessive ROS exposure, Roquin-2 promotes ubiquitination and degradation of TAK1 to suppress Nrf2 activation, and thereby contributes to an efficient cell death, providing insight into the pathogenesis of oxidative stress-related diseases, including cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Utilizing medicinal plants and other natural resources to prevent different types of human cancers is the prime focus of attention. Cervical cancer in women ranks as the fourth most common type of malignancy. The current study used gas chromatography-mass spectrometry (GC-MS) to identify the active phytochemical constituents from Caladium lindenii leaf extracts using ethanol (ECL) and n-hexane (HCL) solvents. Plant extracts were tested for potential cytotoxic effects on HeLa and HEK-293 T cells using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) and the crystal violet assays. SYBR Green-based real-time PCR was performed to assess the mRNA expression profile of the apoptosis biomarkers (BCL-2 and TP53). The molecular interaction of the compounds with the targeted proteins (TP53, BCL2, EGFR, and HER2) was determined using molecular docking. GC-MS analysis revealed a total of 93 compounds in both extracts. The ECL extract significantly reduced the proliferation of HeLa cervical cancer cells, with an IC 50 value of 40 µg/mL, while HEK-293 T cells showed less effect (IC 50  = 226 µg/mL). The quantitative RT-PCR gene expression analysis demonstrated the ethanol extract regulated TP53 and BCL2 mRNA expressions in treated cancer cell samples. Heptanediamide, N,N'-di-benzoyloxy-(- 10.1) is the best-docked ligand with a TP53 target found in the molecular docking study, whereas EGFR/Clionasterol had the second highest binding affinity (- 9.7), followed by EGFR/Cycloeucalenol (- 9.6). It is concluded that ECL extract has promising anti-cervical cancer potential and might be valued for developing new plant-derived anticancer agents after further investigations., (© 2024. The Author(s).)

Ploidy, p53, bcl-2, and c-myc genes are associated with gastric cancer. Myc target 1 (MYCT1) gene is an oncogenic gene and is associated with cancer progression through different signal transduction pathways identifying the corresponding genes The objective of the study was to evaluate the association between MYCT1 gene expression and gastric cancer. Real-time polymerase chain reaction (RT-PCR), western blot analysis, cell growth study, and TUNEL assay were performed for the human gastric cancer cell lines and human embryonic kidney cell line. β-Actin gene preferred as a control in RT-PCR. The ratio of MYCT1 gene expression to β-actin gene expression less than 0.5 was considered as downregulation. Using SDS-PAGE MYCT1 gene expression was measured in western blot analysis. Cells with and without the MYCT1 gene were incubated in 35 mm plates with 10% fetal bovine serum in the cell growth study. TUNEL assay was performed to detect the effect of the MYCT1 gene on the apoptosis of cells. The ratio of MYCT1 gene expression to β-actin gene expression was 0.47 ± 0.01 and 0.76 ± 0.01 for human gastric cancer cell lines and human embryonic kidney cell lines, respectively. MYCT1 gene expression was downregulated in the human gastric cancer cell lines than human embryonic kidney cell line (p < 0.001). MYCT1 gene decreased cell growth (p = 0.041) during 6 days of incubation study of cells. TUNEL assay showed only the fluorescence of PI in BGC823 cells without the MYCT1 gene. MYCT1 gene expression was downregulated in the human gastric cancer cell lines, and MYCT1 gene accelerates the apoptotic process., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The overuse of antibiotics in both humans and livestock has led to the antibiotic resistance phenomenon which is now considered one of the biggest problems in the modern world. Some antibiotics used to control or prevent infections in livestock poultry were registered a long time ago, and as a result, data on the possible side effects of their use, both for birds and humans, are incomplete and should be updated. An example of such an antibiotic is enrofloxacin which has been widely used in poultry since 1989. Data in recent years have begun to indicate that this antibiotic induces the process of apoptosis in diverse types of eukaryotic cells. Unfortunately, such studies have never been conducted on chicken models even though it is in poultry that this antibiotic is most commonly used. Therefore, the purpose of this work was to investigate whether enrofloxacin induces apoptosis in chicken cells of the UMNSAH/DF-1 line and to study the molecular mechanism of its action. The results of these experiments indicated that enrofloxacin induces apoptosis in chicken cells but not in human HEK-293 and PC3 cells. This induction was accompanied by changes in the morphology and size of mitochondria, the process of apoptosome formation and activation of executive caspases, which clearly indicates the role of the mitochondrial pathway in the induction of apoptosis by enrofloxacin. This study is the first to show the toxicity of enrofloxacin against chicken cells and to demonstrate the exact mechanism of its action. The results presented in this work show the need to monitor the concentration of antibiotic residues in poultry foods as well as to study their impact on public health to guarantee consumer safety and prevent the phenomenon of antibiotic resistance in bacteria., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Siderophores are structurally unique medicinal natural products and exhibit considerable therapeutic potential. Herein, we report the design and synthesis of azotochelin, a natural siderophore, and an extensive library of azotochelin analogs and their anticancer properties. We modified the carboxylic acid and the aromatic ring of azotochelin using various chemical motifs. We evaluated the cytotoxicity of the compounds against six different cancer cell lines (KB-3-1, SNB-19, MCF-7, K-562, SW-620, and NCI-H460) and a non-cancerous cell line (HEK-293). Among the twenty compounds tested, the IC 50 values of nine compounds (14, 32, 35-40, and 54) were between 0.7 and 2.0 μM against a lung cancer cell line (NCI-H460). Moreover, several compounds showed good cytotoxicity profile (IC 50 <10 μM) against the tested cancer cell lines. The flow cytometry analysis showed that compounds 36 and 38 induced apoptosis in NCI-H460 in a dose-dependent manner. The cell cycle analysis indicated that compounds 36 and 38 significantly arrested the cell cycle at the S phase to block cancer cell proliferation in the NCI-H460 cell line. The study has produced various novel azotochelin analogs that are potentially effective anticancer agents and lead compounds for further synthetic and medicinal chemistry exploration., (© 2024 Wiley-VCH GmbH.)

Globally, colorectal cancer is the most prevalent type of cancer. Even though multiple treatments such as surgery, radiation, chemotherapy, and immunotherapy are available, the adverse effects caused in patients seem remarkable. Therefore, the current work was deliberated to prepare the metabolites (cell-free supernatant-CFS) from Weissella cibaria RK-3-1 to conduct in-silico and in-vitro-based anticancer assays. First, the active biomolecules present in the CFS were screened using a GC-MS analyzer. In addition, in-silico-based pharmacokinetic and docking studies were performed to confirm the anticancer potential of metabolites. In-silico results suggested that the bioactive compounds such as filicinic acid, dibutyl phthalate, and 4H-pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl present in CFS possessed significant molecular docking interactions with anticancer hub proteins. Furthermore, in-vitro results displayed the inhibition of cell proliferation in HT-29 cells at an IC50 value of 22.5 ± 1.3 µg/ml with the least significant effect on HEK-293 cell lines. Moreover, bacterial metabolites-controlled cell proliferation during the cell cycle's synthesis phase (S). Furthermore, the gene expression results confirm the increased expression of Bad, Bax, Bcl2, caspase-3, and cytochrome-C genes involved in the intrinsic apoptotic pathway. Hence, our findings from the in-silico and the in-vitro study confirm the anticancer potential of cell free-supernatant synthesized by W. cibaria., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The voltage-dependent anion channel 2 (VDAC2) is the abundant protein in the outer mitochondrial membrane. Opening VDAC2 pores leads to the induction of mitochondrial energy and material transport, facilitating interaction with various mitochondrial proteins implicated in essential processes such as cell apoptosis and proliferation. To investigate the VDAC2 in lower vertebrates, we identified Lr-VDAC2, a homologue of VDAC2 found in lamprey (Lethenteron reissneri), sharing a sequence identity of greater than 50 % with its counterparts. Phylogenetic analysis revealed that the position of Lr-VDAC2 aligns with the lamprey phylogeny, indicating its evolutionary relationship within the species. The Lr-VDAC2 protein was primarily located in the mitochondria of lamprey cells. The expression of the Lr-VDAC2 protein was elevated in high energy-demanding tissues, such as the gills, muscles, and myocardial tissue in normal lampreys. Lr-VDAC2 suppressed H 2 O 2 (hydrogen peroxide)-induced 293 T cell apoptosis by reducing the expression levels of Caspase 3, Caspase 9, and Cyt C (cytochrome c). Further research into the mechanism indicated that the Lr-VDAC2 protein inhibited the pro-apoptotic activity of BAK (Bcl-2 antagonist/killer) protein by downregulating its expression at the protein translational level, thus exerting an anti-apoptotic function similar to the role of VDAC2 in humans., (Copyright © 2024. Published by Elsevier Ltd.)

LINC00894 may be associated with synaptic function, but its biology function in neural cells is still unknown. In this study, LINC00894 knockdown decreased the EdU incorporated into newly synthesized DNA and cell viability in MTT or CCK-8 assay in HEK-293T and BE(2)-M17 (M17) neuroblastoma cells. And LINC00894 knockdown increased cellular apoptosis in Annexin V-FITC staining, the expression of activated Caspase3 and the level of reactive oxygen species (ROS) both in HEK-293T and M17 cells. Moreover, LINC00894 also protected cells from hydrogen peroxide induced apoptosis in in vitro models. Utilizing RNA sequencing (RNA-seq) integrated with quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunoblot, we identified that LINC00894 affected activating transcription factor 3 (ATF3) expression in HEK-293T, M17, and SH-SY5Y neuroblastoma cells. Finally, we found that ectopic expression of ATF3 restored cell proliferation and inhibited cell apoptosis in LINC00894 downregulated M17 cells. While knockdown of ATF3 also significantly increased the cell viability inhibition and apoptosis promotion induced by LINC00894 knockdown in M17 cells. Our results from in vitro models revealed that LINC00894 could promote neuronal cell proliferation and inhibit cellular apoptosis by affecting ATF3 expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The endoplasmic reticulum (ER) responds to cellular stress by initiating an unfolded protein response (UPR) that mitigates misfolded protein accumulation by promoting protein degradation pathways. Chronic ER stress leads to UPR-mediated apoptosis and is a common underlying feature of various diseases, highlighting the modulators of the UPR as attractive targets for therapeutic intervention. Ataxia-telangiectasia mutated protein kinase (ATM) is a stress-responsive kinase that initiates autophagy in response to reactive oxygen species (ROS), and ATM deficiency is associated with increased ER stress markers in vitro. However, whether ATM participates in the UPR remains unclear. In this in vitro study, a novel role for ATM in the ER stress response is described using the well-characterized HEK293 cells treated with the common ER stress-inducing agent, tunicamycin, with and without the potent ATM inhibitor, KU-60019. We show for the first time that ATM is activated in a time-dependent manner downstream of UPR initiation in response to tunicamycin treatment. Furthermore, we demonstrate that ATM is required for p62-bound protein cargo degradation through the autophagy pathway in response to ER stress. Lastly, our data suggest a protective role for ATM in ER stress-mediated oxidative stress and mitochondrial apoptosis. Taken together, we highlight ATM as a potential novel drug target in ER stress-related diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Breast cancer is one of the leading causes of cancer deaths worldwide. Thereafter, designing new treatments with higher specificity and efficacy is urgently required. In this regard, targeted immunotherapy using immunotoxins has shown great promise in treating cancer. To target a breast cancer cell, the authors used the antibody fragment against a receptor tyrosine kinase, EphA2, which is overexpressed in many cancers. This fragment was conjugated to a plant toxin, subunit A of ricin, in two different orientations from N to C-terminal (EphA2- C-Ricin and EphA2- N-Ricin). Then, these two immunotoxins were characterized using in vitro cell-based assays. Three different cell lines were treated, MDA-MB-231 (breast cancer) which has a high level of EphA2 expression, MCF-7 (breast cancer) which has a low level of EphA2 expression, and HEK293 (human embryonic kidney) which has a very low level of EphA2 expression. Moreover, binding ability, cytotoxicity, internalization, and apoptosis capacity of these two newly developed immunotoxins were investigated. The flow cytometry using Annexin V- Propidium iodide (PI) method indicated significant induction of apoptosis only in the MDA-MB-231 cells at different concentrations. It was also found that construct I, EphA2- C-Ricin immunotoxin, could bind, internalize, and induce apoptosis better than the EphA2- N-Ricin immunotoxin. In addition, the obtained data suggested that the N or C-terminal orientation conformation is of significant importance., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study assessed OR3 pigment, derived from Streptomyces coelicolor JUACT03, for its anticancer potential on HepG2 liver cancer cells and its safety on HEK293 normal cells. OR3 induced apoptosis and inhibited HepG2 cell proliferation, confirmed by caspase activation, Sub-G1 phase cell cycle arrest, and reduced colony formation. Proteomic analysis revealed altered expression of proteins associated with ribosomal function, mRNA processing, nuclear transport, proteasome activity, carbohydrate metabolism, chaperone function, histone regulation, and vesicle-mediated transport. Downregulation of proteins in MAPKAP kinase1, EIF2, mTOR, and EIF4 pathways contributed to apoptosis and cell cycle arrest. Changes in c-MYC, FUBP1 target proteins and upregulation of Prohibitin-1 (PHB1) were also noted. Western blot analysis supported alterations in eIF2, mTOR, and RAN pathways, including downregulation of RAB 5, c-MYC, p38, MAPK1, and MAPK3. OR3 exhibited significant anti-angiogenic activity in the in ovo CAM assay. In summary, OR3 demonstrated strong anticancer effects, inducing apoptosis, hindering proliferation, and displaying antiangiogenic properties. These findings highlight OR3's potential as an anticancer drug candidate, warranting further in vivo exploration., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Posttranslational modification of proteins by reversible acetylation regulates key biological processes. Histone deacetylases (HDACs) catalyze protein deacetylation and are frequently dysregulated in tumors. This has spurred the development of HDAC inhibitors (HDACi). Such epigenetic drugs modulate protein acetylation, eliminate tumor cells, and are approved for the treatment of blood cancers., Objectives: We aimed to identify novel, nanomolar HDACi with increased potency over existing agents and selectivity for the cancer-relevant class I HDACs (HDAC1,-2,-3,-8). Moreover, we wanted to define how such drugs control the apoptosis-autophagy interplay. As test systems, we used human leukemic cells and embryonic kidney-derived cells., Methods: We synthesized novel pyrimidine-hydroxamic acid HDACi (KH9/KH16/KH29) and performed in vitro activity assays and molecular modeling of their direct binding to HDACs. We analyzed how these HDACi affect leukemic cell fate, acetylation, and protein expression with flow cytometry and immunoblot. The publicly available DepMap database of CRISPR-Cas9 screenings was used to determine sensitivity factors across human leukemic cells., Results: Novel HDACi show nanomolar activity against class I HDACs. These agents are superior to the clinically used hydroxamic acid HDACi SAHA (vorinostat). Within the KH-series of compounds, KH16 (yanostat) is the most effective inhibitor of HDAC3 (IC 50  = 6 nM) and the most potent inducer of apoptosis (IC 50  = 110 nM; p < 0.0001) in leukemic cells. KH16 though spares embryonic kidney-derived cells. Global data analyses of knockout screenings verify that HDAC3 is a dependency factor in 115 human blood cancer cells of different lineages, independent of mutations in the tumor suppressor p53. KH16 alters pro- and anti-apoptotic protein expression, stalls cell cycle progression, and induces caspase-dependent processing of the autophagy proteins ULK1 and p62., Conclusion: These data reveal that HDACs are required to stabilize autophagy proteins through suppression of apoptosis in leukemic cells. HDAC3 appears as a valid anti-cancer target for pharmacological intervention., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)

RNA silencing is a post-transcriptional gene-silencing mechanism mediated by microRNAs (miRNAs). However, the regulatory mechanism of RNA silencing during viral infection is unclear. TAR RNA-binding protein (TRBP) is an enhancer of RNA silencing that induces miRNA maturation by interacting with the ribonuclease Dicer. TRBP interacts with a virus sensor protein, laboratory of genetics and physiology 2 (LGP2), in the early stage of viral infection of human cells. Next, it induces apoptosis by inhibiting the maturation of miRNAs, thereby upregulating the expression of apoptosis regulatory genes. In this study, we show that TRBP undergoes a functional conversion in the late stage of viral infection. Viral infection resulted in the activation of caspases that proteolytically processed TRBP into two fragments. The N-terminal fragment did not interact with Dicer but interacted with type I interferon (IFN) signaling modulators, such as protein kinase R (PKR) and LGP2, and induced ER stress. The end results were irreversible apoptosis and suppression of IFN signaling. Our results demonstrate that the processing of TRBP enhances apoptosis, reducing IFN signaling during viral infection., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Small extracellular vesicles (sEVs) have emerged as promising therapeutic agents and drug delivery vehicles. Targeted modification of sEVs and their contents using genetic modification strategies is one of the most popular methods. This study investigated the effects of p53 fusion with arrestin domain-containing protein 1 (ARRDC1) and CD63 on the generation of sEVs, p53 loading efficiency, and therapeutic efficacy. Overexpression of either ARRDC1-p53 (ARP) or CD63-p53 (CDP) significantly elevated p53 mRNA and protein levels. The incorporation of ARRDC1 and CD63 significantly enhanced HEK293T-sEV biogenesis, evidenced by significant increases in sEV-associated proteins TSG101 and LAMP1, resulting in a boost in sEV production. Importantly, fusion with ARRDC1 or CD63 substantially increased the efficiency of loading both p53 fusion proteins and its mRNA into sEVs. sEVs equipped with ARP or CDP significantly enhanced the enrichment of p53 fusion proteins and mRNA in p53-null H1299 cells, resulting in a marked increase in apoptosis and a reduction in cell proliferation, with ARP-sEVs demonstrating greater effectiveness than CDP-sEVs. These findings underscore the enhanced functionality of ARRDC1- and CD63-modified sEVs, emphasizing the potential of genetic modifications in sEV-based therapies for targeted cancer treatment.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered swine coronavirus with potential cross-species transmission risk. Although SADS-CoV-induced host cell apoptosis and innate immunity antagonization has been revealed, underlying signaling pathways remain obscure. Here, we demonstrated that infection of SADS-CoV induced apoptosis in vivo and in vitro , and that viral protein NS7a is mainly responsible for SADS-CoV-induced apoptosis in host cells. Furthermore, we found that NS7a interacted with apoptosis-inducing factor mitochondria associated 1 (AIFM1) to activate caspase-3 via caspase-6 in SADS-CoV-infected cells, and enhanced SADS-CoV replication. Importantly, NS7a suppressed poly(I:C)-induced expression of type III interferon (IFN-λ) via activating caspase-3 to cleave interferon regulatory factor 3 (IRF3), and caspase-3 inhibitor protects piglets against SADS-CoV infection in vivo . These findings reveal how SADS-CoV induced apoptosis to inhibit innate immunity and provide a valuable clue to the development of effective drugs for the clinical control of SADS-CoV infection.IMPORTANCEOver the last 20 years, multiple animal-originated coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, have caused millions of deaths, seriously jeopardized human health, and hindered social development, indicating that the study of animal-originated coronaviruses with potential for cross-species transmission is particularly important. Bat-originated swine acute diarrhea syndrome coronavirus (SADS-CoV), discovered in 2017, can not only cause fatal diarrhea in piglets, but also infect multiple human cells, with a potential risk of cross-species transmission, but its pathogenesis is unclear. In this study, we demonstrated that NS7a of SADS-CoV suppresses IFN-λ production via apoptosis-inducing factor mitochondria associated 1 (AIFM1)-caspase-6-caspase-3-interferon regulatory factor 3 (IRF3) pathway, and caspase-3 inhibitor (Z-DEVD-FMK) can effectively inhibit SADS-CoV replication and protect infected piglets. Our findings in this study contribute to a better understanding of SADS-CoV-host interactions as a part of the coronaviruses pathogenesis and using apoptosis-inhibitor as a drug as potential therapeutic approaches for prevention and control of SADS-CoV infection., Competing Interests: The authors declare no conflict of interest.

Fas-associated protein with death domain (FADD), procaspase-8, and cellular FLICE-inhibitory proteins (cFLIP) assemble through death-effector domains (DEDs), directing death receptor signaling towards cell survival or apoptosis. Understanding their three-dimensional regulatory mechanism has been limited by the absence of atomic coordinates for their ternary DED complex. By employing X-ray crystallography and cryogenic electron microscopy (cryo-EM), we present the atomic coordinates of human FADD-procaspase-8-cFLIP complexes, revealing structural insights into these critical interactions. These structures illustrate how FADD and cFLIP orchestrate the assembly of caspase-8-containing complexes and offer mechanistic explanations for their role in promoting or inhibiting apoptotic and necroptotic signaling. A helical procaspase-8-cFLIP hetero-double layer in the complex appears to promote limited caspase-8 activation for cell survival. Our structure-guided mutagenesis supports the role of the triple-FADD complex in caspase-8 activation and in regulating receptor-interacting protein kinase 1 (RIPK1). These results propose a unified mechanism for DED assembly and procaspase-8 activation in the regulation of apoptotic and necroptotic signaling across various cellular pathways involved in development, innate immunity, and disease., (© 2024. The Author(s).)

The use of magnetic metal nanoparticles has been considered in cancer treatment studies. In this study, BiFe 2 O 4 @Ag nanoparticles were synthesized biologically by Scenedesmus obliquus for the first time and their anticancer mechanism in a gastric cancer cell line was characterized. The physicochemical properties of the nanoparticles were evaluated by fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic Light Scattering (DLS), and zeta potential analyses. Cell viability and nuclear damage were investigated by the MTT and Hoechst staining assays, respectively. Flow cytometry analysis was performed to determine the frequency of the necrotic and apoptotic cells as well as cell cycle analysis of the nanoparticles-treated cells. Physicochemical characterization showed that the synthesized particles were spherical, without impurities, in a size range of 38-83 nm, with DLS size and zeta potential of 295.7 nm and -27.7 mV, respectively. BiFe 2 O 4 @Ag nanoparticles were considerably more toxic for the gastric cancer cells (AGS cell line) than HEK293 normal cells with IC 50 of 67 and 117 µg/ml, respectively. Treatment of AGS cells with the nanoparticles led to a remarkable increase in the percentage of late apoptosis (38.5 folds) and cell necrosis (13.4 folds) and caused cell cycle arrest, mainly at the S phase. Also, nuclear fragmentation and apoptotic bodies were observed in the gastric cancer cells treated with the nanoparticles. This study represents BiFe 2 O 4 @Ag as a novel anticancer candidate against gastric cancer that can induce cell apoptosis through DNA damage and inhibition of cell cycle progression., (© 2024. The Author(s).)

BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial channel protein VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, while interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK and inhibited BAK apoptotic activity. Thus, this VDAC2-BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease or to promote BAK-mediated apoptosis for cancer therapy., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yuan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

An important epigenetic component of tyrosine kinase signaling is the phosphorylation of histones, and epigenetic readers, writers, and erasers. Phosphorylation of protein arginine methyltransferases (PRMTs), have been shown to enhance and impair their enzymatic activity. In this study, we show that the hyperactivation of Janus kinase 2 (JAK2) by the V617F mutation phosphorylates tyrosine residues (Y149 and Y334) in coactivator-associated arginine methyltransferase 1 (CARM1), an important target in hematologic malignancies, increasing its methyltransferase activity and altering its target specificity. While non-phosphorylatable CARM1 methylates some established substrates (e.g. BAF155 and PABP1), only phospho-CARM1 methylates the RUNX1 transcription factor, on R223 and R319. Furthermore, cells expressing non-phosphorylatable CARM1 have impaired cell-cycle progression and increased apoptosis, compared to cells expressing phosphorylatable, wild-type CARM1, with reduced expression of genes associated with G2/M cell cycle progression and anti-apoptosis. The presence of the JAK2-V617F mutant kinase renders acute myeloid leukemia (AML) cells less sensitive to CARM1 inhibition, and we show that the dual targeting of JAK2 and CARM1 is more effective than monotherapy in AML cells expressing phospho-CARM1. Thus, the phosphorylation of CARM1 by hyperactivated JAK2 regulates its methyltransferase activity, helps select its substrates, and is required for the maximal proliferation of malignant myeloid cells., (© 2024. The Author(s).)

Background: Cancer is the second leading cause of death in the world. Colorectal cancer is the third leading cause of cancer death. Today, there are several options for treating colorectal cancer such as chemotherapy, surgery, radiotherapy, immunotherapy, and gene therapy. 5-Fluorouracil is known as a suitable candidate for the treatment of various cancers, especially colorectal cancer. However, the use of this drug is limited, so it is usually used in combination with other drugs and agents. Based on the evidence obtained, this study attempted to evaluate the combined effects of 5-fluorouracil and caffeine on colorectal cancer cells., Methods: In this study, initially HCT116 and HEK293 cell lines were cultured as cancer and normal cells, respectively. These cell lines were then evaluated for cytotoxicity, induction of apoptosis, and rate of cell migration. All data were analyzed by statistical methods., Results: The results indicated that a combination of caffeine and 5-FU augmented their cytotoxicity in HCT116 cells but reduced cytotoxicity in HEK293 cells. No reduction was observed in the migration of HCT116 cells that were treated with caffeine or a combination of caffeine and 5-FU. Also, it seems that caffeine reverses the apoptotic effect of 5-FU in HCT116 cells.

Background: Rn7SK, a highly conserved small nuclear non-coding RNA, controls Polymerase II transcription machinery by activating of the Positive Transcriptional Elongation Factor b (P-TEFb). Apart from its role in transcriptional regulation, the potential functions of Rn7SK in cell apoptosis are poorly understood. In a previous study, we demonstrated that overexpression of 7SK induces apoptosis in HEK cells. However, it remains unclear whether 7SK-mediated apoptosis induction is exerted through the intrinsic or extrinsic pathways., Methods and Results: Rn7SK was overexpressed in HEK 293T cell line using Lipofectamine 2000 reagent to investigate its potential apoptotic functions. The overexpression of Rn7SK resulted in reduced cell viability through the induction of apoptosis, as evidenced by MTT assay and Annexin V/PI staining. Concurrently, alterations in the expression levels of key apoptosis-related genes were observed, as determined by quantitative RT-PCR. Furthermore, Rn7SK overexpression led to a decrease in cell proliferation, as assessed by colony formation assay and growth curve analysis. This reduction was associated with downregulated expression of key proliferative-related genes. Additionally, the migration and invasion capabilities of cells were significantly inhibited upon upregulation of Rn7SK, as demonstrated by transwell assays., Conclusions: This study suggests the apoptotic role of 7SK through both intrinsic and extrinsic pathways, necessitating further investigation into its underlying mechanisms., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment involving disruptions to inner hair cells (IHCs), ribbon synapses, spiral ganglion neurons (SGNs), and/or the auditory nerve itself. The outcomes of cochlear implants (CI) for ANSD are variable and dependent on the location of lesion sites. Discovering a potential therapeutic agent for ANSD remains an urgent requirement. Here, 293T stable transfection cell lines and patient induced pluripotent stem cells (iPSCs)-derived auditory neurons carrying the apoptosis inducing factor (AIF) p.R422Q variant were used to pursue a therapeutic regent for ANSD. Nicotinamide adenine dinucleotide (NADH) is a main electron donor in the electron transport chain (ETC). In 293T stable transfection cells with the p.R422Q variant, NADH treatment improved AIF dimerization, rescued mitochondrial dysfunctions, and decreased cell apoptosis. The effects of NADH were further confirmed in patient iPSCs-derived neurons. The relative level of AIF dimers was increased to 150.7 % (P = 0.026) from 59.2 % in patient-neurons upon NADH treatment. Such increased AIF dimerization promoted the mitochondrial import of coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4), which further restored mitochondrial functions. Similarly, the content of mitochondrial calcium ( m Ca 2+ ) was downregulated from 136.7 % to 102.3 % (P = 0.0024) in patient-neurons upon NADH treatment. Such decreased m Ca 2+ levels inhibited calpain activity, ultimately reducing the percentage of apoptotic cells from 30.5 % to 21.1 % (P = 0.021). We also compared the therapeutic effects of gene correction and NADH treatment on hereditary ANSD. NADH treatment had comparable restorative effects on functions of ANSD patient-specific cells to that of gene correction. Our findings offer evidence of the molecular mechanisms of ANSD and introduce NADH as a potential therapeutic agent for ANSD therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

STING is a well-known signaling adaptor essential for sensing cytosolic dsDNA to produce type I interferon. Although the detailed underlying mechanisms remain enigmatic, recent studies show that STING activation can lead to T lymphocyte apoptosis. Here, we report that AIFM1 facilitates STING activation-induced cell apoptosis in T lymphocytes. Mechanistically, AIFM1 is upregulated after STING activation in T cells but not in HEK293T-STING and THP-1 cells, rendering T cells more sensitive to apoptosis. In contrast to the canonical role of AIFM1 in the caspase-independent parthanatos, the function of AIFM1 is operated by the formation of an AIFM1/IRF3/BAX complex and mitochondrial outer membrane permeabilization, which cause cytochrome c release and caspase activation. Furthermore, supplementation with newly synthesized AIFM1 can reconstitute STING activation-induced cell apoptosis in HEK293T-STING and THP-1 cells. Our study identifies AIFM1 as a key regulating factor determining the hypersensitivity of T lymphocytes to STING activation-induced cell apoptosis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Nanoplastics (NPs) pollution poses a huge threat to the ecosystem and has become one of the environmental pollutants that have attracted much attention. There is increasing evidence that both oxidative stress and endoplasmic reticulum stress (ERS) are associated with polystyrene nanoplastics (PS-NPs) exposure. Lipopolysaccharide (LPS) has been shown to induce apoptotic damage in various tissues, but whether PS-NPs can aggravate LPS-induced apoptosis in mouse kidneys through oxidative stress-regulated inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) ERS pathway remains unclear. In this study, based on the establishment of in vitro and in vivo PS-NPs and LPS exposure models alone and in combination in mice and HEK293 cells, the effects and mechanisms of PS-NPs on LPS-induced renal cell apoptosis were investigated. The results showed that PS-NPs could aggravate LPS-induced apoptosis. PS-NPs/LPS can induce ERS through oxidative stress, activate the IRE1/XBP1 pathway, and promote the expression of apoptosis markers (Caspase-3 and Caspase-12). Kidney oxidative stress, ERS, and apoptosis in PS-NPs + LPS combined exposure group were more severe than those in the single exposure group. Interestingly, 4-phenylbutyric acid-treated HEK293 cells inhibited the expression of the IRE1/XBP1 ERS pathway and apoptotic factors in the PS-NPs + LPS combined exposure group. N-acetyl-L-cysteine effectively blocked the activation of the IRE1/XBP1 ERS pathway, suggesting that PS-NPs-induced oxidative stress is an early event that triggers ERS. Collectively, these results confirmed that PS-NPs aggravated LPS-induced apoptosis through the oxidative stress-induced IRE1/XBP1 ERS pathway. Our study provides new insights into the health threats of PS-NPs exposed to mammals and humans., (© 2022 Wiley Periodicals LLC.)

Caspases are a family of evolutionary conserved cysteine proteases that play key roles in programmed cell death and inflammation. Among the methods for the detection of caspase activity, biosensors based on luciferases have advantages in genetical encoding and convenience in assay. In this study, we constructed a new set of caspase biosensors based on NanoLuc luciferase. This kind of sensors, named NanoLock, work in dark-to-bright model, with the help of a NanoLuc quencher peptide (HiBiT-R/D) mutated from HiBiT. Optimized NanoLock responded to proteases with high signal to noise ratio (S/N), 1233-fold activation by tobacco etch virus protease in HEK293 cells and > 500-fold induction to caspase 3 in vitro. We constructed NanoLocks for the detection of caspase 1, 3, 6, 7, 8, 9, and 10, and assays in HEK293 cells demonstrated that these sensors performed better than commercial kits in the aspect of S/N and convenience. We further established a cell line stably expressing NanoLock-casp 6 and provided a proof-of-concept for the usage of this cell line in the high throughput screening of caspase 6 modulator., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The purpose of this study was to determine the cytotoxicity of Lactiplantibacillus plantarum strain RD1 (Lpb RD1), which was isolated and identified from the curd by 16 S rRNA sequencing. The probiotic properties of the isolated strain were studied by bile and NaCl tolerance and the ethyl acetate extract of Ea-LpRD1, was used to determine the toxicity against human breast cancer (MCF-7) cell lines and human embryonic kidney (HEK-293) cell lines by MTT assay. DNA fragmentation assay was carried out to study apoptosis induction. Flow cytometry analysis was done to determine the % of a cell population using the FTIC-Annexin V staining method. RT-PCR was used to assess gene expression levels in both cell lines. The IC 50 concentration of the Ea-LpRD1 in MCF-7 cells was 0.30 mg/ml and in HEK-293 was 0.47 mg/ml. The expression levels of the BCL-2 gene anti-apoptotic genes in humans were reduced and BAX, caspase-8, caspase-3, and caspase-9 were an increased expression in MCF-7 cell lines., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Zoledronic acid, a nitrogen-containing bisphosphonate drug, is used for the treatment of osteoporosis, Paget's disease of bone, and tumor-induced osteolysis. Zoledronic acid has also gained a place in cancer treatment due to its cytotoxic and antiproliferative effects in many cancer cells. Although zoledronic acid is considered safe, kidney damage is still one of the concerns in therapeutic doses. In the study, the aim was to assess the nephrotoxic profiles of zoledronic acid in the human embryonic kidney (HEK-293) cells. Cytotoxicity evaluation was performed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and neutral red uptake tests, while oxidative stress was performed by reactive oxygen species (ROS) production via flow cytometry, and the incomprehensible evaluation of ROS-related genes by RT-PCR and apoptosis was performed with Annexin-PI analysis in flow cytometry. The obtained result showed that zoledronic acid inhibited cell viability (IC 50 values were determined as 273.16  by MTT) and cell proliferation in a concentration-dependent manner, induced ROS production, caused glutathione depletion, and increased oxidative stress index and endoplasmic reticulum (ER) stress, indicating severe cellular stress. The expression levels of oxidative damage (L-fabp, α-GST, Nrf2, and HMOX1), ER stress (CASP4, IRE1-α, GADD153, and GRP78), and apoptosis (Bcl-2, Bax, Cyt-c, p53, CASP9, CASP3, NF-κB, TNF-α, and JNK) related genes were altered as well as IRE1-α protein levels. Herein, we were the first to show that increased oxidative stress and ER stress resulting in apoptosis are the key molecular pathways in zoledronic acid-induced nephrotoxicity equivalent to clinically administered concentrations., (© 2022 Wiley Periodicals LLC.)

Heavy-metal pollution has increasingly jeopardized the habitats of marine organisms including the sea cucumber, a seafloor scavenger vital to seawater bio-decontamination, ocean de-acidification and coral-reef protection. Normal physiology including immune functions of sea cucumbers is toxicologically modulated by marine metal pollutants such as cadmium (Cd). The processes underpinning Cd's toxic effects on immune systems in the sea cucumber, Holothuria leucospilota , are still poorly understood. To this end, we cloned and characterized a full-length caspase-9 ( Hl-CASP9 ) cDNA in the sea cucumber, Holothuria leucospilota . Hl-CASP9 mRNA levels evolved dynamically during embryonic development. Coelomocytes, a type of phagocytic immune effectors central to H. leucospilota immunity, were found to express Hl-CASP9 mRNA most abundantly. Hl-CASP9 protein structurally resembles caspases-2 and -9 in both invertebrate and vertebrate species, comprising a CARD domain and a CASc domain. Remarkably, Hl-CASP9 was transcriptionally sensitive to abiotic oxidative stress inducers including hydrogen peroxide (H 2 O 2 ), nitric oxide ( • NO) and cadmium (Cd), but insensitive to immunostimulants including lipopolysaccharide (LPS), and poly(I:C). Overexpression of Hl-CASP9 augmented mitochondria-dependent apoptosis in HEK293T cells, while knock-down of Hl-CASP9 blunted Cd-induced coelomocyte apoptosis in vivo . Overall, we illustrate that an evolutionarily ancient caspase-9-dependent pathway exists to sensitize coelomocytes to premature cell death precipitated by heavy metal pollutants, with important implications for negative modulation of organismal immune response in marine invertebrates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Chen, Wu, Li, Zhang, Jiang, Luo, Hu, Wong and Ren.)

Apoptosis is a form of programmed cell death that is essential for maintaining internal environmental stability. Disordered apoptosis can cause a variety of diseases; therefore, sensing apoptosis can provide help in study of mechanism of the relevant diseases and drug development. It is known that caspase-3 is a key enzyme involved in apoptosis and the expression of its activity is an indication of apoptosis. Here, we present a genetically encoded switch-on mNeonGreen2-based molecular biosensor. mNeonGreen2 is the brightest monomeric green fluorescent protein. The substrate of caspase-3, DEVD amino acid residues, is inserted in it, while cyclized by insertion of Nostoc punctiforme DnaE intein to abolish the fluorescence (inactive state). Caspase-3-catalyzed cleavage of DEVD linearizes mNeonGreen2 and rebuilds the natural barrel structure to restore the fluorescence (activated state). The characterization exhibited that the Caspase-3 biosensor has shortened response time, higher sensitivity, and prolonged functional shelf life in detection of caspase-3 amongst the existing counterparts. We also used the Caspase-3 biosensor to evaluate the effect of several drugs on the induction of apoptosis of HeLa and MCF-7 tumor cells and inhibition of Zika virus invasion., (© 2021. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

p53, master transcriptional regulator of the genotoxic stress response, controls cell-cycle arrest and apoptosis following DNA damage. Here, we identify a p53-induced lncRNA suicidal PARP-1 cleavage enhancer (SPARCLE) adjacent to miR-34b/c required for p53-mediated apoptosis. SPARCLE is a ∼770-nt, nuclear lncRNA induced 1 day after DNA damage. Despite low expression (<16 copies/cell), SPARCLE deletion increases DNA repair and reduces DNA-damage-induced apoptosis as much as p53 deficiency, while its overexpression restores apoptosis in p53-deficient cells. SPARCLE does not alter gene expression. SPARCLE binds to PARP-1 with nanomolar affinity and causes apoptosis by acting as a caspase-3 cofactor for PARP-1 cleavage, which separates PARP-1's N-terminal (NT) DNA-binding domain from its catalytic domains. NT-PARP-1 inhibits DNA repair. Expressing NT-PARP-1 in SPARCLE-deficient cells increases unrepaired DNA damage and restores apoptosis after DNA damage. Thus, SPARCLE enhances p53-induced apoptosis by promoting PARP-1 cleavage, which interferes with DNA-damage repair., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

With the outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), coronaviruses have begun to attract great attention across the world. Of the known human coronaviruses, however, Middle East respiratory syndrome coronavirus (MERS-CoV) is the most lethal. Coronavirus proteins can be divided into three groups: nonstructural proteins, structural proteins, and accessory proteins. While the number of each of these proteins varies greatly among different coronaviruses, accessory proteins are most closely related to the pathogenicity of the virus. We found for the first time that the ORF3 accessory protein of MERS-CoV, which closely resembles the ORF3a proteins of severe acute respiratory syndrome coronavirus and SARS-CoV-2, has the ability to induce apoptosis in cells in a dose-dependent manner. Through bioinformatics analysis and validation, we revealed that ORF3 is an unstable protein and has a shorter half-life in cells compared to that of severe acute respiratory syndrome coronavirus and SARS-CoV-2 ORF3a proteins. After screening, we identified a host E3 ligase, HUWE1, that specifically induces MERS-CoV ORF3 protein ubiquitination and degradation through the ubiquitin-proteasome system. This results in the diminished ability of ORF3 to induce apoptosis, which might partially explain the lower spread of MERS-CoV compared to other coronaviruses. In summary, this study reveals a pathological function of MERS-CoV ORF3 protein and identifies a potential host antiviral protein, HUWE1, with an ability to antagonize MERS-CoV pathogenesis by inducing ORF3 degradation, thus enriching our knowledge of the pathogenesis of MERS-CoV and suggesting new targets and strategies for clinical development of drugs for MERS-CoV treatment., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In order to investigate for a new effective and safe anticancer drug, we synthesized a novel series of quinazoline containing biologically active substituted-sulfonamide moiety at 3- position 4a - n . The structure of the newly prepared compounds was proved by microanalysis, IR, 1 H-NMR, 13 C-NMR and mass spectral data. All the synthesized compounds were evaluated for their in vitro cytotoxic activity in numerous cancer cell lines including A549, HepG-2, LoVo and MCF-7 and normal HUVEC cell line. The two most active compounds 4d and 4f were then tested for their apoptosis induction using DNA content and Annexin V-FITC/PI staining. Moreover, apoptosis initiation was also confirmed using RT-PCR and Western blot. To further understand the binding preferences of quinazoline sulfonamides, docking simulations were used. Among the fourteen new synthesized compounds, we found that compounds 4d and 4f exerted the strongest cytotoxicity against MCF-7 cells with an IC 50 value of 2.5 and 5 μM, respectively. Flow cytometry data revealed the ability of compounds 4d and 4f to mediate apoptosis and arrest cell cycle growth at G1 phase. Furthermore, RT-PCR and Western blot results suggested that both 4d and 4f activates apoptotic cell death pathway in MCF-7 cells. Molecular docking assessments indicated that compounds 4d and 4f fit perfectly into Bcl2's active site. Based on the biological properties, we conclude that both compounds 4d and 4f could be used as a new type of anticancer agent, which provides a scientific basis for further research into the treatment of cancer.

Tripartite motif protein 21 (TRIM21) is an interferon-inducible E3 ligase, containing one RING finger domain, one B-box motif, one coiled-coil domain at the N-terminal, as well as one PRY domain and one SPRY domain at the C-terminal. TRIM21 is expressed in many tissues and plays an important role in systemic autoimmunity. However, TRIM21 plays different roles in different virus infections. In this study, we evaluate the relationship between porcine TRIM21 and PCV2 infection as well as host immune responses. We found that PCV2 infection modulated the expression of porcine TRIM21. TRIM21 can enhance interferons and proinflammatory factors and decrease cellular apoptosis in PCV2-infected cells. These results indicate that porcine TRIM21 plays a critical role in enhancing PCV2 infection, which is a promising target for controlling and developing the treatment of PCV2 infection.

Liver injury is the first step in causing fibrosis, cirrhosis, and liver cancer, leading to mortality. However, the drivers of progressive liver injury are still incompletely defined. Here, we identify GBP5 as a major factor causing liver injury and inflammation. We show that the expression of GBP5 is abnormally elevated in the damaged liver, and its expression depends at least partially on the NF-κB-inducing kinase (NIK)/NF-κB2 signaling pathway. Knockout of Gbp5 ameliorates D-galactosamine/lipopolysaccharide (GalN/LPS)-induced liver injury and inflammation. Conversely, liver-specific overexpression of GBP5 induces liver injury and inflammation. Mechanistically, GBP5 induces hepatocyte apoptosis through the activation of both calpain/caspase 12/caspase 3 and TNFα/caspase 8/caspase 3 signaling pathways. Inhibition of either calpain activity or caspase 3 prevents GBP5-induced cell death. Our data demonstrate that GBP5 expression is induced by toxins or the NIK signaling pathway, which promotes both extrinsic and intrinsic apoptosis signaling pathways and further induces liver injury, providing a novel drug target for the treatment of liver injury and inflammation., (© 2021 Federation of American Societies for Experimental Biology.)

Background: It is well established that ecdysteroid hormones play an important role in arthropod development and reproduction, mediated by ecdysteroid receptors. Ticks are obligate hematophagous arthropods and vectors of pathogens. The salivary gland plays an essential role in tick growth and reproduction and in the transmission of pathogens to vertebrate hosts. During tick development, the salivary gland undergoes degeneration triggered by ecdysteroid hormones and activated by apoptosis. However, it is unknown how the ecdysteroid receptor and apoptosis regulate salivary gland degeneration. Here, we report the functional ecdysteroid receptor (a heterodimer of the ecdysone receptor [EcR] and ultraspiracle [USP]) isolated from the salivary gland of the tick Rhipicephalus haemaphysaloides and explore the molecular mechanism of ecdysteroid receptor regulation of salivary gland degeneration., Methods: The full length of RhEcR and RhUSP open reading frames (ORFs) was obtained from the transcriptome. The RhEcR and RhUSP proteins were expressed in a bacterial heterologous system, Escherichia coli. Polyclonal antibodies were produced against synthetic peptides and were able to recognize recombinant and native proteins. Quantitative real-time PCR and western blot were used to detect the distribution of RhEcR, RhUSP, and RhCaspases in the R. haemaphysaloides organs. A proteomics approach was used to analyze the expression profiles of the ecdysteroid receptors, RhCaspases, and other proteins. To analyze the function of the ecdysteroid receptor, RNA interference (RNAi) was used to silence the genes in adult female ticks. Finally, the interaction of RhEcR and RhUSP was identified by heterologous co-expression assays in HEK293T cells., Results: We identified the functional ecdysone receptor (RhEcR/RhUSP) of 20-hydroxyecdysone from the salivary gland of the tick R. haemaphysaloides. The RhEcR and RhUSP genes have three and two isoforms, respectively, and belong to a nuclear receptor family but with variable N-terminal A/B domains. The RhEcR gene silencing inhibited blood-feeding, blocked engorgement, and restrained salivary gland degeneration, showing the biological role of the RhEcR gene in ticks. In the ecdysteroid signaling pathway, RhEcR silencing inhibited salivary gland degeneration by suppressing caspase-dependent apoptosis. The heterologous expression in mammalian HEK293T cells showed that RhEcR1 interacts with RhUSP1 and induces caspase-dependent apoptosis., Conclusions: These data show that RhEcR has an essential role in tick physiology and represents a putative target for the control of ticks and tick-borne diseases., (© 2021. The Author(s).)

New and potent agents that evade multidrug resistance (MDR) and inhibit epigenetic modifications are of great interest in cancer drug development. Here, we describe that a moniliformin derivative (IUPAC name: 3-(naphthalen-2-ylsulfanyl)-4-{[(2Z)-1,3,3-trimethyl-2,3-dihydro-1H-indol-2-ylidene]methyl}cyclobut-3-ene-1,2-dione; code: MCC1381) bypasses P-gp-mediated MDR. Using transcriptomics, we identified a large number of genes significantly regulated in response to MCC1381, which affected the cell cycle and disturbed cellular death and survival. The potential targets of MCC1381 might be histone deacetylases (HDACs) as predicted by SwissTargetPrediction. In silico studies confirmed that MCC1381 presented comparable affinity with HDAC1, 2, 3, 6, 8 and 11. Besides, the inhibition activity of HDACs was dose-dependently inhibited by MCC1381. Particularly, a strong binding affinity was observed between MCC1381 and HDAC6 by microscale thermophoresis analysis. MCC1381 decreased the expression of HDAC6, inversely correlated with the increase of acetylated HDAC6 substrates, acetylation p53 and α-tubulin. Furthermore, MCC1381 arrested the cell cycle at the G 2 /M phase, induced the generation of reactive oxygen species and collapse of the mitochondrial membrane potential. MCC1381 exhibited in vivo anti-cancer activity in xenografted zebrafish. Collectively, MCC1381 extended cytotoxicity towards P-gp-resistant leukemia cancer cells and may act as a pan-HDACs inhibitor, indicating that MCC1381 is a novel candidate for cancer therapy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Ischemic heart disease in children may be induced by varied factors, and there is no corresponding systematic treatment up to now. This study aims to investigate the effects of microRNA (miR)-148 on myocardial injury in immature rats with myocardial ischemia-reperfusion (MI/R) injury. In this study, MI/R model was established by ligating the coronary artery of heart. The results showed that miR-148 alleviated myocardial injury and rescued relevant parameters (mean ventricular systolic blood pressure (MAP), left ventricular systolic blood pressure (LVSP), heart rate (HR), creatine kinase-MB (CK-MB), cTn1 and Mb in immature rats with MI/R injury. Besides, miR-148 improved the immune dysfunction induced by MI/R through increasing the number of interleukin (IL)-10 + cells and reducing the number of inducible nitric oxide synthase (iNOS) + cells. In addition, miR-148 relieved the apoptosis of cardiomyocytes induced by MI/R through inhibiting the expression of Bax and elevating the expression of Bcl-2. Further molecular mechanism indicated that pyruvate dehydrogenase kinase 4 (PDK4) was the downstream target of miR-148, which was further confirmed by dual luciferase reporter assay and related expression detection. Accordingly, silenced PDK4 attenuated cardiac dysfunction, immune disorder and myocardial apoptosis in immature rats and enhanced the ability of antioxidant enzymes. What is more, activated SMAD pathway induced by MI/R injury was then blocked by silenced PDK4. Taken together, our study demonstrated that overexpressed miR-148 relieved cardiac dysfunction, immune disorder and cardiomyocyte apoptosis in immature MI/R rats by PDK4 inhibition, which provided novel targets for MI/R injury treatment.

Gestational diabetes mellitus (GDM), a common complication of pregnancy, harms the health of pregnant women and fetuses. MicroRNAs (miRNAs) dysregulation in placenta is involved in GDM. Herein, we explored the roles of miR-362-5p in GDM. After high glucose (HG) treated HTR-8/SVneo cells, CCK-8 and flow cytometry were conducted to assess the capability of the proliferation and apoptosis, respectively. The data demonstrated that HG inhibited proliferation and induced apoptosis of HTR-8/SVneo cells. MiR-362-5p level was reduced in HG-treated cells and placenta tissues of GDM patients, measured by qPCR. Overexpressed miR-362-5p accelerated the proliferation and restrained apoptosis of HG-treated cells. Furthermore, glutathione-disulfide reductase (GSR) was verified as a target of miR-362-5p, through TargetScan database and dual-luciferase reporter assay. GSR was upregulated in GDM placenta tissues and was negatively regulated by miR-362-5p. Enforced GSR level abolished the effects of miR-362-5p overexpression on the proliferation and apoptosis of HTR-8/SVneo cells. Furthermore, miR-362-5p increased p-PI3K, p-AKT and bcl-2, while reduced bax and cleaved caspase3, which were abolished by GSR. In conclusion, miR-362-5p promoted cell proliferation and inhibited apoptosis via targeting GSR and activating PI3K/AKT pathway. The findings mentioned above suggested that miR-362-5p might be a therapy target of GDM.

Two compounds (7- O -methylmearnsitrin (7-OM) and roseoside A (RA) were identified and characterized from the leaves of Leea aequata ( L . aequata ) L. The cytotoxicity of 7-OM and RA on HeLa cells was performed using MTT. The 7-OM and RA showed significant inhibition of HeLa cell proliferation with an IC 50 of 22 and 20 µg/mL, respectively when compared with the standard vincristin sulphate (VS) (IC 50 of 15 µg/mL). Moreover, the 7-OM and RA significantly inhibit other cancer cells (HEK-293, H228, and H3122) when compared with the VS and the cytotoxic activity of the compounds might show through the induction of apoptosis. Strikingly, annexin-V and PI signals could barely be detected in control cells, while strong fluorescence densities were observed in response to treatment indicating that these compounds have capacity to induce HeLa cell apoptosis. Our results suggest that the anticancer activity of 7-OM and RA was due to the induction of apoptosis.

Spinster homolog 3 (SPNS3) belongs to the Spinster (SPNS) family which participates in sphingolipid transportation through the cell membrane. However, the functions of SPNS3 in acute myeloid leukemia (AML) are unknown. This study obtained SPNS3 from a gene set that was related to AML relapse and evaluate whether high SPNS3 expression induced apoptosis resistance in an AML cell line, which is consistent with the role of SPNS3 as a marker of poor prognosis in the clinic. Moreover, internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-ITD) mutation and the AC127521.1/ MIR-139/SPNS3 competing endogenous RNA axis were found to regulate SPNS3 expression. In addition, we noted that SPNS3 may play an important role in the Sphingosine-1-phosphate signal pathway that is involved in the maintenance of the AML microenvironment. These results highlight the anti-apoptosis effect of SPNS3 in AML, and the potential mechanism mediating this effect was explored through bioinformatics. Abbreviations: AML: acute myeloid leukemia; FLT3-ITD: internal tandem duplication of FMS-like tyrosine kinase 3; SPNS3: spinster homolog 3; SPNS1: spinster homolog 1; SPNS2: spinster homolog 2; GO: gene ontology; S1P: sphingosine-1-phosphate; ceRNA: competing endogenous RNA; dAML: acute myeloid leukemia at diagnosis; iAML: acute myeloid leukemia after induction chemotherapy; rAML: acute myeloid leukemia at relapse; DEGs: differentially expressed genes; BP: biological processes; CC: cellular components; MF: molecular functions; MRD: minimal residual disease; EFS: event-free survival; OS: overall survival; KEGG: Kyoto Encyclopedia of Genes and Genomes; SPHK: Sphingosine kinase.

Lung cancer, which is exacerbated by environmental pollution and tobacco use, has become the most common cause of cancer-related deaths worldwide, with a five-year overall survival rate of only 19% (Siegel et al., 2020; Yang et al., 2020; Yu and Li, 2020). Nearly 85% of lung cancers are non-small cell lung cancers, of which lung adenocarcinoma is the most common subtype accounting for 50% of non-small cell lung cancer cases. At present, radiotherapy is the primary therapeutic modality for lung cancer at different stages, with significant prolongation of survival time (Hirsch et al., 2017; Bai et al., 2019; Shi et al., 2020). Irradiation can generate reactive oxygen species (ROS) through the radiolysis reaction of water and oxygen, cause DNA damage and oxidative stress, and subsequently result in cancer cell death (Kim et al., 2019). Nevertheless, radioresistance seriously hinders the success of treatment for lung cancer, owing to local recurrence and distant metastasis (Huang et al., 2021). Compared with small cell lung cancer, non-small cell lung cancer shows more tolerance to radiotherapy. Therefore, it is of great importance to decipher key mechanisms of radioresistance and identify effective molecular radiosensitizers to improve patient survival.

Damaged or superfluous cells are typically eliminated by apoptosis. Although apoptosis is a cell-autonomous process, apoptotic cells communicate with their environment in different ways. Here we describe a mechanism whereby cells under apoptotic stress can promote survival of neighbouring cells. We find that upon apoptotic stress, cells release the growth factor FGF2, leading to MEK-ERK-dependent transcriptional upregulation of pro-survival BCL-2 proteins in a non-cell autonomous manner. This transient upregulation of pro-survival BCL-2 proteins protects neighbouring cells from apoptosis. Accordingly, we find in certain cancer types a correlation between FGF-signalling, BCL-2 expression and worse prognosis. In vivo, upregulation of MCL-1 occurs in an FGF-dependent manner during skin repair, which regulates healing dynamics. Importantly, either co-treatment with FGF-receptor inhibitors or removal of apoptotic stress restores apoptotic sensitivity to cytotoxic therapy and delays wound healing. These data reveal a pathway by which cells under apoptotic stress can increase resistance to cell death in surrounding cells. Beyond mediating cytotoxic drug resistance, this process also provides a potential link between tissue damage and repair., (© 2021. The Author(s).)

Acetylcholinesterase inhibitors (AChEIs), the most developed treatment strategies for Alzheimer's disease (AD), will be used in clinic for, at least, the next decades. Their side effects are in highly variable from drug to drug with mechanisms remaining to be fully established. The withdrawal of tacrine (Cognex) in the market makes it as an interesting case study. Here, we found tacrine could disrupt the proper trafficking of proline-rich membrane anchor-linked tetrameric acetylcholinesterase (AChE) in the endoplasmic reticulum (ER). The exposure of tacrine in cells expressing AChE, e.g., neurons, caused an accumulation of the misfolded AChE in the ER. This misfolded enzyme was not able to transport to the Golgi/plasma membrane, which subsequently induced ER stress and its downstream signaling cascade of unfolded protein response. Once the stress was overwhelming, the cooperation of ER with mitochondria increased the loss of mitochondrial membrane potential. Eventually, the tacrine-exposed cells lost homeostasis and underwent apoptosis. The ER stress and apoptosis, induced by tacrine, were proportional to the amount of AChE. Other AChEIs (rivastigmine, bis(3)-cognitin, daurisoline, and dauricine) could cause the same problem as tacrine by inducing ER stress in neuronal cells. The results provide guidance for the drug design and discovery of AChEIs for AD treatment. SIGNIFICANCE STATEMENT: Acetylcholinesterase inhibitors (AChEIs) are the most developed treatment strategies for Alzheimer's disease (AD) and will be used in clinic for at least the next decades. This study reports that tacrine and other AChEIs disrupt the proper trafficking of acetylcholinesterase in the endoplasmic reticulum. Eventually, the apoptosis of neurons and other cells are induced. The results provide guidance for drug design and discovery of AChEIs for AD treatment., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Background/aim: The use of iodinated contrast media may impair renal function. However, no report has addressed the nephrotoxicity of high doses of iodinated contrast media in normal kidney cells and its associated molecular mechanisms., Materials and Methods: Cell proliferation was assessed using the MTT assay. Cell death was evaluated through examining the morphological changes and TUNEL assay. Autophagy was detected through acridine orange staining and lysotracker staining. Reactive oxygen species production and AKT kinase activity were examined., Results: Iopromide induced cell death and triggered apoptosis and autophagy in HEK 293 cells. Cell viability was significantly restored in the presence of a pan-caspase inhibitor or a ROS scavenger, N-acetyl-L-cysteine. AKT kinase activity was found to be reduced in iopromide-treated HEK 293 cells., Conclusion: High concentrations of iopromide induce cell damage, apoptosis, and autophagy through down-regulating AKT and ROS-activated cellular stress pathways in HEK 293 cells., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Urinary bladder cancer (UBC) is considered one of the most prevalent malignant tumors worldwide. Complementary and integrative approaches for the treatment of bladder cancer, such as the intake of isoflavonoid phytoestrogens, are of increasing interest due to the risk of mortality and long-term morbidity associated with surgical procedures. The biological effects of prunetin, one of the less-studied phytoestrogens, have not yet been examined in this respect. Therefore, this study aimed to explore the efficacy of prunetin on UBC cells (RT-4). METHODS AND RESULTS: The cytotoxicity and nitric oxide synthase activities of prunetin were determined in cell cultures. The expression of apoptosis-related genes was determined with RT-PCR. Cell cycle assays were performed using a flow cytometer and cellular apoptotic rate was measured. The results suggested that prunetin has cytotoxic effects at 21.11 µg/mL on RT-4 cells. Flow cytometry analysis showed that prunetin induced apoptosis and arrested th cell cycle in the G 0 /G 1 phase. Prunetin exposure was associated with increases in CASP3 and TNF-α gene expression in RT-4 cells at doses of 21.11 and 42.22 µg/mL, respectively. Strong nitric oxide inhibition was observed at IC 50 of 5.18 µg/mL under macrophage mediated inflammatory circumstances., Conclusions: Prunetin possesses anti-cancer properties and may be a candidate compound for the prevention of UBC. This is the first study that evaluated prunetin for its in vitro antitumor activities, clarified its possible apoptotic molecular mechanism and provided novel insights into its anti-inflammatory nature and effects on the expression of related key genes., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways. By employing the SWATH-MS technique, we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes. Combining SWATH-MS-based network modeling and experimental validation, we found that when RIP1 level is below ~1000 molecules/cell (mpc), the cell solely undergoes TRADD-dependent apoptosis. When RIP1 is above ~1000 mpc, pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount, which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical observations that RIP1 is required for necroptosis but its increase down-regulates necroptosis. Higher amount of RIP1 (>~46,000 mpc) suppresses apoptosis, leading to necroptosis alone. The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic. Our study provides a resource for encoding the complexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes, enabling RIP1 to play diverse roles in governing cell fate decisions., (© 2021. The Author(s).)

Cervical cancer is a serious gynecological cancer and one of the primary causes of mortality in female patients with cancer. Despite advances in cancer research, the molecular mechanism underlying cancer remains poorly understood. High levels of MIR9‑3 host gene (HG) are associated with the occurrence and development of cervical cancer. However, the specific role of MIR9‑3HG during the development of cervical cancer is unclear. In the present study, the expression of MIR9‑3HG was silenced in C33A and SiHa cervical cancer cell lines. Proliferation and apoptosis were measured in these cells using 5‑ethynyl‑2'‑deoxyuridine assay and flow cytometry, respectively. In addition, targeting microRNAs (miRs) of MIR9‑3HG and mRNAs of miR‑498 were predicted using public databases. The predicted interactions between these molecules were validated using RNA immunoprecipitation, RNA pull‑down and luciferase reporter assays. Lastly, C33A cells transfected with short hairpin MIR‑3HG alone or in combination with miR‑498 inhibitor or PC‑EP300 were subcutaneously injected into mice. The levels of miR‑498, EP300 and Ki67 in tumor tissue were measured via reverse transcription‑quantitative PCR or western blotting. MIR9‑3HG knockdown inhibited the proliferation of cervical cancer cells, whilst promoting apoptosis. MIR9‑3HG sponged miR‑498 and inhibited its expression. Additionally, miR‑498 interacted with EP300 and inhibited its expression. Transfection with miR‑498 inhibitor significantly decreased apoptosis levels; this effect was abolished following EP300 silencing in vitro . In vivo , both miR‑498 inhibition and EP300 overexpression reversed the inhibition of tumor growth mediated by MIR‑3HG knockdown. MIR9‑3HG promoted the proliferation cervical cancer cells via EP300 and miR‑498. These in vitro and in vivo findings demonstrate the regulatory role of the MIR9‑3HG/miR‑498/EP300 axis in cervical cancer cell growth. Thus, the present study identified novel molecular targets for the diagnosis and treatment of cervical cancer and provided new insight into the pathogenesis of cervical cancer.