Corneal transparency is critical for optimal visual function, and corneal neovascularization represents the primary cause of visual impairment globally. Recent studies have identified the transcription factor BMAL1 as a significant regulator of angiogenesis. However, its specific role and underlying mechanisms in endothelial cell apoptosis remain inadequately understood. This study seeks to elucidate the role and underlying mechanisms of BMAL1 in endothelial cell apoptosis by employing genetic modification, alkali-burned mouse corneal neovascularization models, lentiviral transfection, proteomic analysis, and other complementary methodologies. Our results showed that BMAL1 expression is significantly elevated in corneal neovascularization induced by alkali burn and removal of Bmal1 in endothelial cells resulted in the suppression of corneal neovascularization in alkali burn mouse models. In vivo experiments have demonstrated that the knockout of Bmal1 in endothelial cells leads to an increase in endothelial cell apoptosis. Complementary in vitro studies revealed that overexpression of BMAL1 in endothelial cells inhibits apoptosis, while knockdown of BMAL1 promotes apoptosis. Proteomic analysis identified STAT6 as a downstream target of BMAL1 involved in the regulation of endothelial cell apoptosis. Further cell salvage experiments confirmed that BMAL1 modulates endothelial cell apoptosis through the regulation of STAT6 expression. Finally, the results of dual-luciferase reporter assay demonstrated that BMAL1 exerts transcriptional repressive effects on the promoter bound by STAT6. This study elucidates the novel role and mechanism of BMAL1 in the regulation of angiogenesis and endothelial cell apoptosis, thereby identifying a potential therapeutic target for the treatment of vascular diseases such as corneal neovascularization.

MLL gene rearrangement recurrently occurs in acute myeloid leukemia (MLL-r AML), which is closely associated with chemotherapy insensitivity and unfavorable clinical outcomes. More importantly, there are limited therapeutic options for the management of patients with MLL-r AML, thus necessitating novel effective treatment strategies. In this study, we demonstrated that low doses of triptolide (LD TPL) and the XPO1 inhibitor selinexor exerted synergistic therapeutic effects on poor-outcome MLL-r AML in vitro, ex vivo and in vivo. Induction of mitochondrial outer membrane permeabilization (MOMP) and initiation of the mitochondrial apoptotic pathway were closely involved in the therapeutic synergy of LD TPL in combination with selinexor against MLL-r AML. Mechanistically, MYC downregulation mediated by the Rap1/Raf/MEK/ERK pathway rather than by PI3K/AKT signaling was implicated in the synergistic activity of the combined regimen. In addition, the induction of DNA damage also contributed to the synergistic effects of the combined regimen on MLL-r AML. In summary, our findings suggest that LD TPL in combination with selinexor might represent a promising therapeutic approach for the treatment of MLL-r AML. However, future clinical trials are mandatory to draw a decisive conclusion.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in tumor cells but faces limitations due to resistance mechanisms involving anti-apoptotic regulators such as Bcl-2. This study investigates Tasisulam, a molecular glue degrader, that degrades RNA-binding motif protein 39 (RBM39), as a sensitizer for TRAIL-mediated apoptosis in renal cell carcinoma (RCC). Tasisulam enhances TRAIL-induced apoptosis by activating both extrinsic and intrinsic apoptotic pathways, achieved through upregulation of death receptor 5 (DR5) and downregulation of B-cell lymphoma 2 (Bcl-2). Importantly, Tasisulam selectively sensitizes RCC cells to TRAIL-induced apoptosis without affecting normal cells.RBM39 knockdown mimicked the effects of Tasisulam by upregulating DR5, downregulating Bcl-2, and enhancing TRAIL-induced apoptosis, suggesting RBM39 as a critical regulator of these pathways. To address TRAIL instability in vivo, AaLS/TRAIL nanoparticles were employed in combination with Tasisulam in a Caki-1 xenograft model. This combination significantly reduced tumor volume and weight compared to single treatments, without observed toxicity. These findings demonstrate that Tasisulam sensitizes RCC cells to TRAIL-induced apoptosis through RBM39-dependent DR5 upregulation and Bcl-2 downregulation. This combination strategy holds significant promise as a potential solution to overcoming TRAIL resistance and advancing more effective treatment outcomes for RCC.

Bladder cancer (BC) is a common urological malignancy for which effective treatments are lacking. In recent years, valosin-containing protein (VCP) has emerged as a potential target for the treatment of cancers. CB-5083 is a VCP inhibitor that has been evaluated in phase I clinical trials. However, drug resistance and severe side effects hamper the application of CB-5083. Mounting evidence suggests that combined treatment is a useful strategy to improve anticancer efficiency with lower toxicity. The aim of this study was to evaluate the combined effects of CB-5083 and luteolin (Lut), a natural flavonoid, on BC cells.

Cellular viability was measured via MTT assays. The cell cycle distribution, degree of cell death and mitochondrial membrane potential were assayed via flow cytometry. mRNA levels were assayed via qRT-PCR. Protein levels were measured via western blotting. RNA interference was applied to knockdown genes. Xenograft experiments were conducted to evaluate the toxicity in vivo.

Cotreatment with CB-5083 and luteolin synergistically reduced the viability of BC cells. In addition, cotreatment with CB-5083 and Lut synergistically induced cell cycle arrest at the G1 phase and apoptosis in BC cells. Mechanistically, CB-5083/Lut cooperatively reduced the expression of Bcl-xl and Mcl-1 in BC cells. Moreover, CB-5083 and Lut synergistically induced endoplasmic reticulum (ER) stress in BC cells. The genetic or pharmacological inhibition of ER stress markedly reduced the degree of apoptosis induced by CB-5083, Lut or their combination in BC cells. In addition, combined treatment with CB-5083 and Lut synergistically repressed the growth of BC cells in vivo.

Our data suggest that combined treatment with CB-5083 and Lut might be applied to treat BC.

Temozolomide (TMZ) chemoresistance is a major challenge in the management of glioblastoma (GBM). Marine-derived fungal metabolites are a significant source of potential chemotherapeutic candidates. This study aimed to investigate the cytotoxic effect of MHO7 (6-epi-ophiobolin G) on GBM cells. MHO7 inhibited GBM cell proliferation and promoted apoptosis, accompanied by a reduction in Akt activity and membrane phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) content. We verified that MHO7 could react with phosphatidylethanolamine (PE), the second most abundant phospholipid in the plasma membrane, to form a covalent adduct. Pre-incubation with exogenous PE significantly alleviated the pro-apoptotic effect of MHO7, with a concomitant increase in Akt activity and membrane PIP2 and PIP3 content. Since binding to PIP3 is a key step in Akt activation, our results indicate that MHO7 can function as a novel Akt inhibitor. Additionally, MHO7 has a synergistic pro-apoptotic effect with TMZ, and TMZ-resistant GBM cells remain sensitive to MHO7. MHO7 had little cytotoxicity against normal neuronal cells. The anti-growth effect of MHO7 was also observed in an orthotopic glioma mice model. Therefore, MHO7 is a promising chemotherapeutic agent for GBM. This study also indicated that membrane lipid-targeted therapy may be a novel and effective strategy for tumor treatment.

This study aims to evaluate the anticancer properties of chamomile nano-emulsion (Cha-NE) and α-lactalbumin (α-LA) coated Cha-NE (LA-Cha-NE) against breast tumor through both in vitro and in vivo investigations. Both Cha-NE and LA-Cha-NE exhibited typical semi-spherical forms under Transmission electron microscope (TEM), and displayed surface charges of 46.75 and 28.45 mV with average sizes of 87.46 and 112.75 nm, respectively. In a safe manner, Cha-NE and LA-Cha-NE showed higher selectivity against breast cancer (MDA-MB-231 and MCF7) cells than normal (HSF) cells. Reductions in serum contents of IL1β, TNF-α, IL-4, IL-6, IL-10, ASAT, ALAT, creatinine, urea, triglycerides, and cholesterol, as well as an the administration of LA-Cha-NE, breast tumor incidence dramatically reduced. Thus, improvement in survival rates leads to successful prevention of mammary tumorigenesis as proved by the histopathological and immunohistochemistry findings. However, there was an increase in mammary GSH, GPx, CAT, and SOD activity. Both in vitro and in vivo investigations showed that LA-Cha-NE had a beneficial therapeutic effect, exhibiting more significantly regulated apoptosis and elevated expression of genes that regulate the cell cycle. Thus, this study demonstrated that the chemo-preventive property of LA-Cha-NE may offer a brand-new alternative therapy to cure breast cancer by re-establishing the compromised oxidative stress response, enhancing the immune response, reducing inflammation process, and fortifying the apoptosis pathway.

During the course of viral infections, IFN-I producing pDCs are fundamental in establishing antiviral defense. However, little is known about the molecular mechanisms by which biological sex contributes to differences in IFN-I production by pDCs. Here, we aimed to identify X-chromosome-encoded proteins as a source of sex differences in IFN-I responses by pDCs. We identified the host-cell factor DDX3 as a key mediator for the sex dimorphism in the IFNα response. DDX3 was significantly higher expressed in female pDCs and was translocated together with IRF7 to the nucleus to orchestrate IFN-I transcription. DDX3 as driver of sex differences in the initial and chronic IFN-I response might serve as a novel target to limit IFN-I-mediated hyperactivation of immune cells.

Toona sinensis, a kind of phytochemicals in traditional Chinese medicine widely used in South-East Asia, has been recognized for its anticancer properties, particularly against various types of cancer. We aimed to evaluate the effectiveness of T. sinensis leaf extracts (TSL) specifically for glioblastoma multiforme (GBM). Gallic acid was identified as the major active component in the aqueous extracts of TSL using the HPLC system. Furthermore, it has been shown to have the ability to penetrate the blood-brain barrier. Various concentrations of TSL (10, 20, 40, and 80 μg/mL) were applied and 80 μg/mL TSL treatment significantly inhibited cell growth, proliferation, and cytotoxicity in A172 and U251 GBM cells. Flow cytometry analysis revealed cell cycle arrest at the G2/M phase and increased apoptotic cells. Furthermore, we observed mitochondrial dysfunction characterized by elevated ROS levels and reduced ATP production due to the blockade of electron transport chain (ETC) complexes. TSL treatment regulated this ROS-induced mitochondrial dysfunction. Western blotting analysis showed upregulation of Bax and Puma, along with downregulation of Bcl-2. Additionally, TSL treatment induced the cleavage of caspase-3, caspase-9, and PARP, indicating activation of the mitochondria-mediated apoptosis pathway and caspase-dependent pathway in both GBM cell lines. To investigate the involvement of the MEK/ERK pathway in TSL-induced effects, we used U0126, an inhibitor of MEK/ERK kinase. The results demonstrated that TSL treatment suppressed MEK/ERK activation, inhibiting ROS-induced mitochondrial dysfunction and promoting apoptosis. This suggests a potential therapeutic strategy targeting the MEK/ERK pathway in GBM treatment. Overall, our findings indicate that TSL treatment exerts cytotoxic effects through ROS-mediated mitochondrial dysfunction and activation of apoptotic pathways via MEK/ERK pathway in GBM cells. These insights provide valuable knowledge for potential therapeutic applications of TSL in GBM treatment.

Acute myeloid leukemia (AML) has traditionally been linked to a poor prognosis, particularly in older patients who are ineligible for intensive chemotherapy. The advent of Venetoclax, a powerful oral BH3 mimetic targeting anti-apoptotic protein BCL2, has significantly advanced AML treatment. Its combination with the hypomethylating agent azacitidine (AZA/VEN) has become a standard treatment for this group of AML patients, demonstrating a 65% overall response rate and a median overall survival of 14.7 months, compared to 22% and 8 months with azacitidine monotherapy, respectively. However, resistance and relapses remain common, representing a significant clinical challenge. Recent studies have identified molecular alterations, such as mutations in FLT3-ITD, NRAS/KRAS, TP53, and BAX, as major drivers of resistance. Additionally, other factors, including metabolic changes, anti-apoptotic protein expression, and monocytic or erythroid/megakaryocytic differentiation status, contribute to treatment failure. Clinical trials are exploring strategies to overcome venetoclax resistance, including doublet or triplet therapies targeting IDH and FLT3 mutations; novel epigenetic approaches; menin, XPO1, and MDM2 inhibitors; along with immunotherapies like monoclonal antibodies and antibody-drug conjugates. A deeper understanding of the molecular mechanisms of resistance through single-cell analysis will be crucial for developing future therapeutic strategies.

Cellular senescence is a stress response that cells can employ to resist cell death. Senescent cells rely on anti-apoptotic signaling for their survival, which can be targeted by senolytic agents, like the BCL-XL, BCL-2, BCL-W inhibitor ABT-263. However, the response to ABT-263 of senescent cancer cells ranges from highly sensitive to refractory. Using BH3 profiling, we identify here apoptotic blocks in cancer cells that are resistant to this senolytic treatment and discover a correlation between mitochondrial apoptotic priming and cellular sensitivity to ABT-263 in senescence. Intriguingly, ABT-263 sensitivity correlates with overall mitochondrial apoptotic priming, not only in senescence but also in the parental state. Moreover, we confirm that ABT-263 exposure increases dependency on MCL-1, which is most enhanced in ABT-263 sensitive cells. ABT-263 resistant cells however upregulate MCL-1, while sensitive cells exhibit low levels of this anti-apoptotic protein. Overall, our data indicate that the response of senescent cells to ABT-263 is predetermined by the mitochondrial apoptotic priming state of the parental cells, which could serve as a predictive biomarker for response to senolytic therapy.

Myocardial ischemia-reperfusion injury (MIRI) is fatal to patients, leading to cardiomyocyte death and myocardial remodeling. Reactive oxygen species (ROS) and oxidative stress play important roles in MIRI. There is a complex crosstalk between ROS and regulatory cell deaths (RCD) in cardiomyocytes, such as apoptosis, pyroptosis, autophagy, and ferroptosis. ROS is a double-edged sword. A reasonable level of ROS maintains the normal physiological activity of myocardial cells. However, during myocardial ischemia-reperfusion, excessive ROS generation accelerates myocardial damage through a variety of biological pathways. ROS regulates cardiomyocyte RCD through various molecular mechanisms. Targeting the removal of excess ROS has been considered an effective way to reverse myocardial damage. Many studies have applied antioxidant drugs or new advanced materials to reduce ROS levels to alleviate MIRI. Although the road from laboratory to clinic has been difficult, many scholars still persevere. This article reviews the molecular mechanisms of ROS inhibition to regulate cardiomyocyte RCD, with a view to providing new insights into prevention and treatment strategies for MIRI.

Acute Myeloid Leukemia (AML) remains a formidable clinical challenge, predominantly due to the emergence of resistance to existing therapeutic regimens, including BCL-2 inhibitors like Venetoclax. Here, a novel approach is introduced by engineering BH3 mimetics utilizing non-canonical amino acids (ncAAs) to achieve dual inhibition of MCL-1 and BCL-xL. Through site saturation mutagenesis scanning, the I58(Chg) mutation is identified, significantly enhancing binding affinity with IC50 values of 2.77 nm for MCL-1 and 10.69 nm for BCL-xL, reflecting an increase of fourfold or more. The developed vMIP-II-TAT-I peptide, incorporating a CXCR4-targeted penetrating motif, demonstrated superior cellular uptake, with mean fluorescence intensity (MFI) 7.2-fold higher in CXCR4-positive AML cells and exhibited a high selectivity index (SI) for AML cells, with minimal impact on normal human hematopoietic stem cells (HSCs). When combined with Venetoclax, this peptide induced synergistic apoptosis, reducing tumor burden and prolonging survival in an AML mouse model, with median survival extended to 53 days from 37 days with Venetoclax alone. These findings reveal the therapeutic potential of dual inhibition in overcoming Venetoclax resistance and selectively targeting leukemic cells with reduced off-target effects, while laying the foundation for developing advanced BH3 mimetics with enhanced targeting, binding affinity, and efficacy for AML treatment.

Tumor necrosis factor receptor-associated factor (TRAF) family is a critical signal transduction protein, and plays important roles in cell growth, apoptosis, and immune response, etc. In this study, molecular characteristics, expression patterns, and the role of TRAF2 in blood parrot Vieja synspila ♀ × Amphilophus citrinellus ♂, an important ornamental fish, were explored response to lipopolysaccharide (LPS) challenge. The full length of blood parrot TRAF2 was 2725 bp, with an open reading frame (ORF) of 1551 bp encoding 516 amino acids, and a molecular weight of 58.58 kDa. Blood parrot TRAF2 contained four conserved domains: RING, TRAF-type zinc finger, TRAF_BIRC3_bd, and MATH (Meprin and TRAF-C homology). Analysis of phylogenetic relationships showed that TRAF2 were conserved in different species, indicating that its role might be similar. Blood parrot TRAF2 mRNA could be detected in all of the tissues examined, and was distributed in both the cytoplasm and nucleus. The expression of blood parrot TRAF2 was up-regulated during LPS challenge. Overexpression of TRAF2 could significantly inhibit the activities of nuclear factor κB (NF-κB) and activated protein 1 (AP-1), and reduce the ratio of Bax/Bcl-2. This study indicated that the TRAF2 might play important roles in organisms during pathogen infection.

Rationale: In the era of precision medicine, there is a growing need for rapid reliable ex vivo functional assays capable of predicting treatment efficacy. One drug class that may particularly benefit from such assays is BH3 mimetics. These small molecules antagonize anti-apoptotic proteins such as BCL-2, MCL-1, or BCL-XL, on which cancer cells depend for their survival. A functional assay known as BH3 profiling was previously developed to measure those dependencies through the use of specific BH3-only peptides. A variation of this technique, dynamic BH3 profiling (DBP), allows for measuring changes in those dependencies, after ex vivo treatment with a drug of interest. Though well-validated to predict clinical response in hematologic malignancies, BH3 profiling technique requires the use of specialized BH3-only peptides and requires significant optimization to achieve reproducible results. Methods: We used a toolkit of BH3 mimetics drugs as probes instead of BH3-only peptides. This technique reduces the complexity and cost by using Annexin V/7AAD staining instead of cytochrome c release as a functional readout for apoptosis. We also used cell lines as internal controls for a representative response to BH3 mimetics that allow us to easily compare and stratify patients according to their profile. Results: We demonstrate that our new protocol enables apoptotic dependencies to be measured efficiently across different hematologic malignancies. In addition to a detailed description of the assay, we describe the results in several models including cell lines and primary tumor cells, both at baseline and dynamically after ex vivo drug treatments. We also compared BH3 toolkit baseline results on cell lines with those obtained using conventional BH3 profiling. Conclusion: Overall, our data validates this streamlined BH3 drug toolkit, allowing for a more extensive use of the BH3 profiling technique.

Infections caused by non-tuberculous mycobacteria (NTM), such as Mycobacterium abscessus, elicit diverse cell death mechanisms including apoptosis, necrosis, and pyroptosis, which play key roles in immunopathogenesis. NTM can manipulate these cell death pathways to evade host immune responses, ensuring their intracellular survival and persistence. Apoptosis may aid in antigen presentation and immune activation, while necrosis and pyroptosis trigger excessive inflammation, leading to tissue damage. Autophagy, a crucial cellular defense mechanism, is often induced in response to NTM infection; however, M. abscessus has evolved mechanisms to inhibit autophagic processes, enhancing its ability to survive within host cells. This manipulation of cell death pathways, particularly the dysregulation of autophagy and ferroptosis, contributes to chronic infection, immune evasion, and tissue damage, complicating disease management. Understanding these mechanisms offers potential therapeutic targets for improving treatment strategies against M. abscessus infections.

The clinical application of BCL-2/xL inhibitors for cancer treatment is limited by the on-target thrombocytopenia. Although APG-1252 was designed to mitigate this issue, platelet toxicity at higher doses in clinical trials restricts dose escalation for greater efficacy. We have developed albumin nanocomplexes of APG-1252 (Nano-1252) to reduce platelet toxicity while improving drug efficacy through enhancing drug delivery to lymphoid organs. Nano-1252 forms stable nanoparticles due to the strong binding affinity between APG-1252 and albumin, reducing the platelet toxicity threshold by fourfold by limiting premature drug release and conversion to its active forms in circulation. Furthermore, Nano-1252 exhibited preferential accumulation in lymphoid organs, leading to enhanced anticancer efficacy in Mantle Cell Lymphoma (MCL) and Myeloproliferative Neoplasms (MPNs) mouse models. Our study not only develops a potential formulation to overcome the current translational barrier of APG-1252 but also reveals novel properties of the well-established albumin nanoformulation, thereby expanding its clinical applications.

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder marked by a gradual decline in memory and cognitive functions. It is characterized by the presence of senile plaques, neurofibrillary tangles, and neuronal degeneration, affecting a significant portion of the human population. A key feature of various nervous system disorders, including AD, is extensive cellular death caused by apoptosis, which affects not only neurons but also glial cells. While apoptosis plays a vital role in eliminating certain cells and supporting normal development, alterations or disruptions in apoptotic pathways can lead to harmful neurodegenerative conditions such as AD. Thus, targeting apoptosis presents a promising therapeutic approach for these diseases. MicroRNAs (miRNAs), a class of non-coding RNA, play diverse roles in cellular functions, including proliferation, gene expression regulation, programmed cell death, intercellular communication, and angiogenesis. By modulating regulatory genes, miRNAs can influence apoptosis, either promoting or inhibiting it. Aberrant expression of miRNAs can impact multiple apoptotic pathways, potentially driving the progression of AD and related health issues. This review summarizes recent research on miRNAs and their dual role in exacerbating or protecting against neural cell damage in AD by altering apoptotic pathways. The regulation of apoptosis by miRNAs offers a prospective therapeutic strategy for Alzheimer's disease.

Tumor-specific apoptosis exerts considerable curative efficacy in cancer, particularly with TRAIL, which has been approved in the clinic; however, therapeutic outcome is compromised due to apoptosis evasion and the short half-life of exogenously infused TRAIL. Herein, we propose a synergistic apoptosis strategy of orthotopic TRAIL expression for enhancing the bystander effect and mitochondrial photodamage for intrinsic apoptosis activation. To actualize synergetic apoptosis, we develop cascade-targeting nanoparticles to perform cell-to-mitochondria shuttling, in which TRAIL-expressing plasmid (pTRAIL) is coprecipitated with calcium phosphate on a glycyrrhetinic acid (GA)-modified graphene oxide nanosheet. For apoptosis synergy, GA mediates tumor accumulation of nanoparticles, followed by structure dissociation for efficient pTRAIL release and expression (cascade module I). Thereafter, GA-modified graphene carriers perform mitochondria distribution for laser-triggered photodamage (cascade module II). The nanoparticles yield tumor inhibition of 86.78% in the melanoma model and demonstrate metastasis blocking activity. Collectively, a cascade-targeting apoptosis technology via a combination of TRAIL-specific bystander effects and mitochondrial photodamage provides innovative oncotherapy synergy.

Many biological signaling pathways employ proteins that competitively dimerize in diverse combinations. These dimerization networks can perform biochemical computations in which the concentrations of monomer inputs determine the concentrations of dimer outputs. Despite their prevalence, little is known about the range of input-output computations that dimerization networks can perform and how it depends on network size and connectivity. Using a systematic computational approach, we demonstrate that even small dimerization networks of 3-6 monomers are expressive, performing diverse multi-input computations. Further, dimerization networks are versatile, performing different computations when their protein components are expressed at different levels, such as in different cell types. Remarkably, individual networks with random interaction affinities, when large enough, can perform nearly all potential one-input network computations merely by tuning their monomer expression levels. Thus, even the simple process of competitive dimerization provides a powerful architecture for multi-input, cell-type-specific signal processing.

Mitochondrial outer membrane permeabilization (MOMP) refers to the increase in permeability of the mitochondrial outer membrane, allowing proteins, DNA, and other molecules to pass through the intermembrane space into the cytosol. As a crucial event in the induction of apoptosis, MOMP plays a significant role in regulating various forms of cell death, including apoptosis, ferroptosis, and pyroptosis. Importantly, MOMP is not a binary process of "all-or-nothing." Under sub-lethal stress stimuli, cells may experience a phenomenon referred to as minority MOMP (miMOMP), where only a subset of mitochondria undergo functional impairment, thereby disrupting the normal life cycle of the cell. This can lead to pathological and physiological changes such as tumor formation, cellular senescence, innate immune dysfunction, and chronic inflammation. This review focuses on the diversity of MOMP events to elucidate how varying degrees of MOMP under different stress conditions influence cell fate. Additionally, it summarizes the current research progress on novel antitumor therapeutic strategies targeting MOMP in clinical contexts.

Over the recent years, our understanding of the cell death machinery of mature erythrocytes has been greatly expanded. It resulted in the discovery of several regulated cell death (RCD) pathways in red blood cells. Apoptosis (eryptosis) and necroptosis of erythrocytes share certain features with their counterparts in nucleated cells, but they are also critically different in particular details. In this review article, we summarize the cell death subroutines in the erythroid precursors (apoptosis, necroptosis, and ferroptosis) in comparison to mature erythrocytes (eryptosis and erythronecroptosis) to highlight the consequences of organelle clearance and associated loss of multiple components of the cell death machinery upon erythrocyte maturation. Recent advances in understanding the role of erythrocyte RCDs in health and disease have expanded potential clinical applications of these lethal subroutines, emphasizing their contribution to the development of anemia, microthrombosis, and endothelial dysfunction, as well as their role as diagnostic biomarkers and markers of erythrocyte storage-induced lesions. Fas signaling and the functional caspase-8/caspase-3 system are not indispensable for eryptosis, but might be retained in mature erythrocytes to mediate the crosstalk between both erythrocyte-associated RCDs. The ability of erythrocytes to switch between eryptosis and necroptosis suggests that their cell death is not a simple unregulated mechanical disintegration, but a tightly controlled process. This allows investigation of eventual pharmacological interventions aimed at individual cell death subroutines of erythrocytes.

Breast cancer remains a significant challenge in oncology, despite notable advances in treatment methods. Traditional therapies such as surgery, chemotherapy, radiation, and hormonal treatments have long been used to manage breast cancer. However, often patients experience treatment failure, resulting in disease recurrence and progression. Therefore, this study explores the therapeutic potential of green-synthesized silver nanoparticles (AgNPs), using the root methanol (MeOH) extract of the African medicinal plant Dicoma anomala (D. anomala) as a reducing agent, to combat breast cancer. AgNPs were synthesized using a bottom-up approach and later modified with liposomes (Lip) loaded with the photosensitizer zinc phthalocyanine tetrasulfonate (Lip@ZnPcS4) through the thin film hydration method. Prior to in vitro cell culture studies, UV-Vis spectroscopy was used to study the in vitro drug release kinetics of nanoparticles (NPs) at pH 5.8 and 7.4 respectively. After a 24 h treatment period, MCF-7 breast cancer cells were evaluated for cell cytotoxicity using lactate dehydrogenase Cyto-Tox96® Non-Radioactive Cytotoxicity Assay Kit LDH and cell viability using the CellTiter-Glo® ATP luminescence assay kit. Cell death studies were analyzed using an inverted light microscope for morphological changes, fluorescence microscopy for reactive oxygen species (ROS) detection and Live/Dead cell viability, human p53 protein analysis using enzyme-linked immunosorbent assay (ELISA), apoptotic and anti-apoptotic protein analysis by immunofluorescence, and gene expression analysis using real-time reverse transcription polymerase chain reaction (RT-PCR) assay. The experiments were conducted in quadruplicate (n = 4), and the results were analyzed using IBM SPSS statistical software version 27, with a 95 % confidence interval. The synthesized NPs and nanocomplexes, including AgNPs, AgNPs-Lip, Lip@ZnPcS4, and AgNPs-Lip@ZnPcS4, demonstrated significant cytotoxicity and therapeutic potential against MCF-7 breast cancer cells. Notably, apoptosis was induced, primarily through the activation of the intrinsic pathway. Given the difficult prognosis associated with breast cancer, these findings highlight the promise of liposomal nanoformulations (NFs) in cancer photodynamic therapy (PDT), supporting further investigation in in vivo settings.

The current study investigated differences in ethanol-induced apoptosis of spinal cord dorsal horn neurons at different developmental stages and the molecular mechanisms involved. A mouse ethanol intervention model was established on postnatal days 4, 7, and 12. Primary cells were derived from the spinal cord at postnatal day 4. Western blotting, immunofluorescence, and flow cytometry were used to detect apoptosis-related proteins in the spinal cord and primary cells. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes originating from the Gene Expression Omnibus dataset GSE184615 was conducted. Effects on Akt/GSK3β pathway proteins were investigated using the GSK3β inhibitor AR-A014418, and the Akt inhibitor DHA. Lentiviral knockdown and overexpression of intervening GSK3β were used in HT22 cell lines to investigate the effects of alcohol on GSK 3β and caspase proteins. J-aggregates, reactive oxygen species assays, and calcein-AM assays were used to investigate mitochondrial function and cell viability. Ethanol caused downregulation of Akt activity and upregulation of GSK3β activity and apoptosis. DHA, AR-A014418, and knockdown of GSK3β effectively counteracted ethanol-induced apoptosis, whereas overexpression of GSK3β enhanced the injury process. PI3K activity was unchanged during these processes. Fluorescence colocalization analysis indicated that BAX was translocated to mitochondria during the apoptotic process. BAX was downregulated as the spinal cord developed, consistent with a reduced susceptibility to ethanol-induced apoptosis. Akt/GSK3β signaling and BAX together determine the direction of alcohol-induced apoptosis and its susceptibility to change during developmental stages in the spinal cord.

Retinoic acid-inducible gene I (RIG-I) is a critical sensor of viral RNA and is activated in response to binding to RNA containing exposed 5'-triphosphate (5'ppp) and poly-uridine to trigger innate immune activation and response including induction of type I and III interferons (IFNs). RIG-I signaling plays a key role in not only restricting RNA virus infection but also suppressing tumor progression via oncolytic signaling. We evaluated the actions of a specific RIG-I agonist RNA (RAR) as a potential therapeutic against model tumor cell lines representing hepatocellular carcinoma (HCC). RAR constitutes a synthetic-modified RNA motif derived from the hepatitis C virus genome that is specifically recognized by RIG-I and induces innate immune activation when delivered to cells. We found that RAR directs RIG-I-dependent signaling to drive HCC cell death. Analysis of knockout cell lines lacking RIG-I, mitochondrial activator of virus signaling, or IRF3 confirmed that RAR-induced cell death signaling propagates through the RIG-I-like receptor (RLR) pathway to mediate caspase activation and HCC cell death. RAR-induced cell death is potentiated by type I IFN. Thus, RAR actions trigger HCC cell death through RIG-I linkage of RLR, caspase, and IFN signaling programs. RAR offers a potent application in antitumor therapeutic strategies leveraging innate immunity against liver cancer.

Aim: This study investigates the correlation between indoxyl sulfate (IS) levels and cognitive impairment in end-stage renal disease (ESRD) patients from human study, in vivo and in vitro study. Materials and Methods: Comparison of demographic and biochemical data, including IS concentrations, was conducted between a control group(n=16) and the ESRD with cognitive impairment group (n=14) and without cognitive impairment (n=17). A CKD animal model induced renal impairment in adenine-fed C57BL/6 mice, assessing memory loss and behavioral changes. Immunohistochemistry evaluated choline acetyltransferase activity and GFAP expression. Differentiating SH-SY5Y cells were treated with IS, assessing cell viability and apoptosis via annexin V and propidium iodide staining and western blotting. Reactive oxidized species generation was measured using DCFCA fluorescence and NAC pretreatment. Results: In ESRD patients with cognitive impairment, IS levels were significantly higher compared to healthy controls, along with older age. CKD mice exhibited renal impairment and memory loss, accompanied by altered choline acetyltransferase activity and GFAP expression. IS treatment induced early apoptosis in SH-SY5Y cells, associated with increased cleaved caspase 3 levels and Fas/Fas-ligand activity, altered Bax/Bcl2 ratio, and reactive oxidized species generation. Conclusion: Elevated IS levels are associated with cognitive impairment and neuronal apoptosis, potentially mediated by oxidative stress. IS could be a therapeutic target for cognitive dysfunction in CKD, necessitating further research into its mechanisms and therapeutic interventions.

Juvenile myelomonocytic leukemia (JMML) is caused by constitutively activated RAS signaling and characterized by increased proliferation and predominant myelomonocytic differentiation of hematopoietic cells. Using MxCre;Ptpn11D61Y/+ mice, which model human JMML, we show that RAS pathway activation affects apoptosis signaling through cell type-dependent regulation of BCL-2 family members. Apoptosis resistance observed in monocytes and granulocytes was mediated by overexpression of the anti-apoptotic and down-regulation of the pro-apoptotic members of the BCL-2 family. Two anti-apoptotic proteins, BCL-XL and MCL-1, were directly regulated by the oncogenic RAS signaling but, in addition, were influenced by microenvironmental signals. While BCL-XL and BCL-2 were required for the survival of monocytes, MCL-1 was essential for neutrophils. Interestingly, stem and progenitor cells expressing the oncogenic PTPN11 mutant showed no increased apoptosis resistance. BCL-XL inhibition was the most effective in killing myeloid cells in vitro but was insufficient to completely resolve myeloproliferation in vivo.

The BH3-interacting domain death agonist (Bid), a proapoptotic signaling molecule of the B-cell lymphoma 2 (Bcl-2) family, is a key regulator of mitochondrial outer membrane (MOM) permeability. Uniquely positioned at the intersection of extrinsic and intrinsic apoptosis pathways, Bid links death receptor signaling to the mitochondria-dependent cascade and can also be activated by endoplasmic reticulum (ER) stress. In its active forms, cleaved Bid (cBid) and truncated Bid (tBid), it disrupts MOM integrity via Bax/Bak-dependent and independent mechanisms. Apoptosis plays a dual role in viral infections, either promoting or counteracting viral propagation. Consequently, viruses modulate Bid signaling to favor their replication. The deregulation of Bid activity contributes to oncogenic transformation, inflammation, immunosuppression, neurotoxicity, and pathogen propagation during various viral infections. In this work, we explore Bid's structure, function, activation processes, and mitochondrial targeting. We describe its role in apoptosis induction and its involvement in infections with multiple viruses. Additionally, we discuss the therapeutic potential of Bid in antiviral strategies. Understanding Bid's signaling pathways offers valuable insights into host-virus interactions and the pathogenesis of infections. This knowledge may facilitate the development of novel therapeutic approaches to combat virus-associated diseases effectively.

Machado-Joseph disease, also known as spinocerebellar ataxia type 3 (MJD/SCA3), is a fatal autosomal dominant hereditary ataxia characterized by cerebellar ataxia resulting from the abnormal expansion of CAG repeats in exon 10 of the ATXN3 gene. At present, there is no effective treatment for SCA3. Small interfering RNAs (siRNAs) are emerging as potential therapeutic strategies to target the disease-causing mutant ATXN3 (mATXN3) protein specifically. However, the efficiency of delivery of siRNAs remains a major obstacle for clinical application, particularly in brain disorders. The aim of this study was to develop a synthetic biology strategy to reprogram the host liver as a tissue chassis to induce and deliver in vivo self-assembled siRNAs to target the ATXN3 gene. A synthetic construct directed by a cytomegalovirus promoter was designed to encode a neuron-targeting rabies virus glycoprotein tag and mATXN3-siRNA. After intravenous injection, the synthetic construct was taken up by mouse livers, which were then reprogrammed to enable the self-assembly, production and secretion of small extracellular vesicles encapsulating mATXN3-siRNA. The small extracellular vesicle-encapsulated mATXN3-siRNA was transported through the endogenous circulating system of small extracellular vesicles, crossing the blood-brain barrier and reaching the cerebellar cortex and spinal cerebellar tract, where they silenced the ATXN3 gene. Treatment with the synthetic construct for 8 or 12 weeks led to significant improvements in motor balance ability and reduction of cerebellar atrophy in YACMJD84.2 transgenic mice. The number of Purkinje cells in the cerebellar cortex was significantly increased, and the loss of myelin basic protein was reduced. Moreover, the quantity of neurotoxic nuclear inclusion bodies and the expression of glial fibrillary acidic protein, which promotes neuroinflammation in activated astrocytes, were decreased significantly. The synthetic construct facilitated the generation and delivery of in vivo self-assembled siRNA to the cerebellar cortex and spinal cerebellar tract, thereby inhibiting the expression of mATXN3 protein. This treatment successfully addressed motor impairments, alleviated neuropathological phenotypes and mitigated neuroinflammation in YACMJD84.2 transgenic mice. Our strategy effectively overcomes the primary challenges associated with siRNA therapy for cerebellar ataxia, offering a promising avenue for future clinical treatments.

Current treatments for acute myeloid leukemias (AMLs) cure fewer than 30 % of patients. This low efficacy is due, in part, to the inter-patient and intra-patient heterogeneity of AMLs; accordingly, all current AML treatment regimens involve drug combinations (polypharmacy). A recently-completed clinical trial in relapsed/refractory AML using a combination of two newer targeted antileukemics, the BCL-2 inhibitor venetoclax (VEN) plus the FLT3 inhibitor gilteritinib (GIL), yielded highly promising results for this two-drug polypharmacy combination. Polypharmacology - wherein a single drug molecule that inhibits two or more biological targets is created - has been proposed to offer superior therapeutic results, as compared to the corresponding polypharmacy approach. Herein, we designed and synthesized several polypharmacologic dual BCL-2/FLT3 hybrid single-molecule inhibitors by tethering VEN to GIL, through their solvent-exposed domains. While the in vitro antileukemic activity of the two-drug VEN + GIL polypharmacy combination proved superior to our focused library of VEN-GIL hybrids, alternative grafting points on GIL may yield improved results for future hybrid compounds.

Parkinson's disease is a neurodegenerative proteinopathy that primarily affects mesencephalic dopaminergic neurons. This dopaminergic depletion can be phenotypically reproduced in various experimental models through the administration of two neurotoxins: N-methyl-4-phenylpyridinium (MPP+) and 6-hydroxydopamine (6-OHDA). The mechanisms underlying the cell death processes induced by these toxins remain a subject of debate. In this context, studies suggest that oxidative-stress-related processes may contribute to the dysfunction and death of dopaminergic neurons. Therefore, investigating pharmacological compounds that can counteract these processes remains crucial for developing therapeutic strategies targeting these neuropathological mechanisms. Withania somnifera (L.) Dunal, commonly known as ashwagandha, is a plant whose roots are used in Ayurvedic medicine to treat various ailments, including those affecting the central nervous system. The active compound Withaferin-A (WFA), a steroid lactone from the withanolide group, is reported to possess antioxidant properties. In this study, we explored the potential neuroprotective effects of WFA and two of its molecular derivatives, cr-591 and cr-777, which contain, respectively, an additional cysteine or glutathione chemical group, known for their antiradical properties. We demonstrated that WFA and its two derivatives, cr-591 and cr-777, protect the integrity and function of dopaminergic neurons exposed to the neurotoxins MPP+ and 6-OHDA both in vitro, using primary mesencephalic neuron cultures from rodents, and in vivo, using the nematode Caenorhabditis elegans.

Tay-Sachs disease is a neurodegenerative disorder characterized by a build-up of GM2 ganglioside in the brain, which results in progressive central nervous system dysfunction. Our group recently generated Hexa-/-Neu3-/- mice, a murine model with neuropathological abnormalities similar to the infantile form of Tay-Sachs disease. Previously, we reported progressive neurodegeneration with neuronal loss in the brain sections of Hexa-/-Neu3-/- mice. However, the relationship between the severity of neurodegeneration and the imbalance in redox homeostasis was not yet clarified in Hexa-/-Neu3-/- mice. Here, we evaluated whether neurodegeneration is associated with oxidative stress in the tissues and cells of Hexa-/-Neu3-/- mice and neuroglia cells from Tay-Sachs patients.

Cell death and oxidative stress-related markers were evaluated in four brain regions and fibroblasts of 5-month-old WT, Hexa-/-, Neu3-/-, and Hexa-/-Neu3-/- mice and human neuroglia cells using Western blot, RT-PCR, and immunohistochemistry analyses. We further analyzed oxidative stress levels in the samples using flow cytometry analyses. We discovered neuronal death, alterations in intracellular ROS levels, and damaging effects of oxidative stress, especially in the cerebellum and fibroblasts of Hexa-/-Neu3-/- mice.

Our results showed that alteration in redox homeostasis might be related to neurodegeneration in the murine model of Tay-Sachs Disease. These findings suggest that targeting the altered redox balance and increased oxidative stress might be a rational therapeutic approach for alleviating neurodegeneration and treating Tay-Sachs disease.

Herein, we developed a dual-factor recognition activatable probe, S1-F, which accurately lights up Bcl-2 and GSH dual-overexpressed cancer tissue with a superior tumor-to-normal tissue (T/N) ratio, highlighting the potential for this probe to be used in the visual diagnosis and imaging-guided surgery of cancer.

Cell death is a fundamental process that needs to be maintained to balance cellular functions and prevent disease. There are several cell death pathways; however, apoptosis and parthanatos are the most prominent and have important roles in cancer biology. As an extremely well-regulated process, apoptosis removes damaged or abnormal cells via caspase activation and mitochondrial involvement. Unlike in the healthy cells, the loss of ability to induce apoptosis in cancer permits tumor cells to survive and multiply out of control and contribute to tumor progression and therapy resistance. On the contrary, parthanatos is a caspase-independent metabolic collapse driven by poly (ADP-ribose) polymerase 1 (PARP1) overactivation, translocation of apoptosis-inducing factor (AIF), and complete DNA damage. Several cancer models are involved with parthanatos. Deoxypodophyllotoxin (DPT) induces parthanatos in glioma cells by excessive ROS generation, PARP1 upregulation, and AIF nuclear translocation. Like in acute myeloid leukemia (AML), the cannabinoid derivative WIN-55 triggers parthanatos, and the effects can be reversed by PARP inhibitors such as olaparib. Developing cancer treatment strategies involving advanced cancer treatment strategies relies on the interplay between apoptosis and parthanatos. However, such apoptosis-based cancer therapies tend to develop resistance, so there is an urgent need to look into alternative pathways like parthanatos, which may not always trigger apoptosis. In overcoming apoptosis resistance, there is evidence that combining apoptosis-inducing agents, such as BH3 mimetics, with PARP inhibitors synergistically enhances cell death. Oxidative stress modulators have been found to promote the execution of parthanatic and apoptotic pathways and allow treatment. In this review, apoptosis and parthanatos are thoroughly compared at the molecular level, and their roles in cancer pathogenesis as related to cancer therapeutic potential are discussed. We incorporate recent findings to demonstrate that not only can parthanatos be used to manage therapy resistance and enhance cancer treatment via the combination of parthanatos and apoptosis but also that immunity and bone deposition can feasibly be employed against long-circulating cancer stem cells to treat diverse forms of metastatic cancers.

Characterizing protein-ligand interactions is crucial to understanding cellular metabolism and guiding drug discovery and development. Herein, we explore complementing hydrogen/deuterium exchange mass spectrometry (HDX-MS) with a recently developed Fenton chemistry-based approach to protein oxidative footprinting mass spectrometry (OX-MS) to discriminate the binding of small-molecule therapeutics. Using drug-dependent perturbation as the experimental report, this combination of techniques more clearly differentiates the in-solution binding profiles of Venetoclax (ABT-199, GDC-0199-AbbVie and Genentech) and a drug candidate S55746 (Servier) to the apoptotic regulatory protein Bcl-2 than either technique alone. These results highlight the value of combining these methods to compare compounds in drug discovery and development. To better understand the structural context of the HDX-MS and OX-MS drug-dependent perturbations, we mapped these data on Bcl-2-Venetoclax and Bcl-2-S55746 cocrystal structures and compared these results with the structure of apo Bcl-2. HDX-MS shows that Venetoclax more strongly impacts the protein backbone compared to S55746. OX-MS reveals oxidation perturbations rationalized by direct side-chain protection as well as by crystallographically observed drug-induced protein restructuring. Both methods report the perturbation of some, but not all, residues mapped within 4 Å of the bound drugs in the crystal structures. Concordant characterization of backbone and side-chain accessibility will enhance our understanding of in-solution protein structure dynamics and protein-ligand interactions during drug discovery, development, and characterization, particularly when high-resolution structures are lacking.

Neuroblastoma (NB) is a type of paediatric cancer that originates from embryonic sympathoadrenal cells. Despite its paediatric origin, NB is mostly treated with strategy of non-small cell lung cancer like adults due to lack of specific therapeutic approach. To improve treatment outcome for NB patients, developing drugs that specifically target the genetic mutations or molecular pathways involved in neuroblastoma is necessary. Overexpression of the insulin-like growth factor 1 receptor (IGF1R) has been linked to various malignancies, including paediatric cancers. We hypothesized that inhibiting IGF1R with ALK (NB specific mutation) by phytochemical compound could effectively treat NB while avoiding undesirable cytotoxic effects. We evaluated the efficacy of Picropodophyllotoxin (PPP) as IGF1R inhibitor, for treatment of NB. The IC50 value of PPP on SH-SY5Y, NB cells after 24 h of treatment was found to be 0.501 μM. Molecular docking studies revealed that PPP had a binding score of -7.5 kcal/mol with IGF1R and - 8.8 kcal/mol with ALK. This suggests that PPP not only binds to and inhibits IGF1R but also has a strong affinity for ALK. Gene expression studies, densitometric analysis, scratch assays, and AO/EtBr differential staining were used to evaluate the efficacy of PPP in NB cells. Transcript expression and densitometric analysis revealed that PPP could downregulate IGF1R and ALK in NB cells. Downregulation of SNAIL, a mesenchymal marker, and upregulation of E-cadherin, an epithelial marker, indicated a mesenchymal to epithelial transition in NB cells, suggesting that PPP treatment inhibited NB cell migration and proliferation. This was further supported by scratch assay results in our study. Furthermore, gene expression analysis of p53, BAX and BCL2 indicated that PPP induces apoptosis in NB cells. AO/EtBr differential staining revealed apoptotic phenomena in NB cells after 24 h of PPP treatment. Although further research is needed to explore the receptor targeting approach using PPP for IGF1R and ALK inhibition.

Human immunodeficiency virus (HIV) infection continues to be a major global health challenge, affecting 38.4 million according to the Joint United Nations Program on HIV/AIDS (UNAIDS) at the end of 2021 with 1.5 million new infections. New HIV infections increased during the 2 years after the COVID-19 pandemic. Understanding the intricate cellular processes underlying HIV pathogenesis is crucial for developing effective therapeutic strategies. Among these processes, autophagy and programmed cell death modalities, including apoptosis, necroptosis, pyroptosis, and ferroptosis, play pivotal roles in the host-virus interaction dynamics. Autophagy, a highly conserved cellular mechanism, acts as a double-edged sword in HIV infection, influencing viral replication, immune response modulation, and the fate of infected cells. Conversely, apoptosis, a programmed cell death mechanism, is a critical defense mechanism against viral spread and contributes to the depletion of CD4+ T cells, a hallmark of HIV/AIDS progression. This review aims to dissect the complex interplay between autophagy and these programmed cell death modalities in HIV-induced pathogenesis. It highlights the molecular mechanisms involved, their roles in viral persistence and immune dysfunction, and the challenges posed by the viral reservoir and drug resistance, which continue to impede effective management of HIV pathology. Targeting these pathways holds promise for novel therapeutic strategies to mitigate immune depletion and chronic inflammation, ultimately improving outcomes for individuals living with HIV.

Background: Osteoporosis has become an inevitable health issue with global aging, and the current drug treatments often have adverse side effects, highlighting the need for safer and more effective therapies. Collagen-derived peptides are promising alternatives due to their favorable safety profile and biological activity. This study aimed to investigate the osteogenic and anti-apoptotic properties of collagen peptide UU1 (GASGPMGPR) in addition to its antioxidant activity. Methods: The effects of UU1 were evaluated in MC3T3-E1 cells by assessing osteogenic markers, including alkaline phosphatase (ALP), Cyclin D1, runt-related transcription factor 2 (Runx2), and Akt/β-catenin signaling. Western blot analysis quantified collagen I, osteocalcin, and phosphorylated Akt levels. Anti-apoptotic effects were measured via p-Akt levels and the Bax/Bcl-2 ratio. Computational molecular docking was performed to explore the molecular mechanism of UU1 via its interaction with epidermal growth factor receptor (EGFR) and collagen-binding integrin. Results: UU1 treatment promoted cell differentiation, with elevated ALP, Cyclin D1, Runx2, and Akt/β-catenin signaling. Notably, at 0.025 mg/mL, UU1 upregulated the levels of collagen I, osteocalcin, and phosphorylated Akt by 2.14, 3.37, and 1.95 times, respectively, compared to the control. Additionally, UU1 exhibited anti-apoptotic effects, indicated by increased p-Akt levels and a reduced Bax/Bcl-2 ratio. Molecular docking analysis suggested that UU1 could assist the dimerization of EGFR, facilitating downstream signaling transductions and activating collagen-binding integrin. Conclusions: These findings highlight UU1 as a multifunctional peptide with antioxidant, osteogenic, and anti-apoptotic properties, positioning it as a promising candidate for anti-osteoporosis applications in the food and pharmaceutical industries.