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The ability to remotely control the activity of chimeric antigen receptors (CARs) with small molecules can improve the safety and efficacy of gene-modified T cells. Split ON- or OFF-switch CARs involve the dissociation of tumor-antigen binding from T cell activation (i.e., CD3ζ) on the receptor (R-) and signaling (S-) chains, respectively, that either associate or are disrupted in the presence of a small molecule. Here, we have developed an inducible (i)ON-CAR comprising the anti-apoptotic B cell lymphoma protein 2 protein in the ectodomain of both chains which associate in the presence of venetoclax. We showed that inducible ON (iON)-CAR T cells respond to target tumors cells in the presence of venetoclax or the BH3 mimetic navitoclax in a dose-dependent manner, while there is no impact of the drugs on equivalent second generation-CAR T cells. Within 48 h of venetoclax withdrawal, iON-CAR T cells lose the ability to respond to target tumor cells in vitro as evaluated by Interferon-gamma (IFNγ) production, and they are reliant upon the presence of venetoclax for in vivo activity. Finally, by fusing a degron sequence to the endodomain of the iON-CAR S-chain we generated an all-in-one ON/OFF-switch CAR, the iONØ-CAR, down-regulated by lenalidomide within 4 to 6 for functionally inactive T cells (no IFNγ production) within 24 h. We propose that our remote-control CAR designs can reduce toxicity in the clinic. Moreover, the periodic rest of iON and iONØ-CAR T cells may alleviate exhaustion and hence augment persistence and long-term tumor control in patients., Competing Interests: Competing interests statement:Provisional intellectual property filing has been filed for iON and iONØ CAR designs with inventors including G.M.P.G.A., B.E.C., SS., and M.I.

Background: Malignant gliomas exhibit rapid tumor progression and resistance to treatment, leading to high lethality. One of the causes is the reduced progression of apoptosis in glioma cells. Layilin is a type 1 transmembrane protein with a C-type lectin motif in its extracellular domain. We previously reported that layilin is mainly localized to mitochondria or their close proximity and that layilin is essential for maintaining of the fragmented type of mitochondria. This study investigates the effects of layilin on mitochondria-mediated apoptosis, focusing on B cell lymphoma (BCL)-2 family proteins in a glioma cell line of A172 cells., Results: We compared the levels of pro-apoptotic BCL-2 family proteins of BAD, BAK, BAX, and BIM and anti-apoptotic BCL-2 family proteins of BCL-2 and BCL-X L between layilin- knockdown (KD) cells and control cells using western blot. The protein levels of BAD were significantly smaller in layilin-KD cells than in control cells, while those of BCL-2 were significantly larger. We then compared the mitochondrial membrane potential (ΔΨm) under p-trifluoromethoxyphenyl hydrazone (FCCP)-treated conditions using MT-1 staining. In layilin-KD cells, ΔΨm was significantly larger and FCCP-induced ΔΨm reduction was significantly lower than in control cells. Furthermore, we examined the levels of cell membrane-bound Annexin V and DNA-bound propidium idodide (PI) in layilin-KD cells with/without staurosporine (STS) treatment. Layilin-KD significantly decreased levels of cell membrane-bound Annexin V with/without STS treatment. On the other hand, PI levels were not changed by layilin-KD. We also investigated the amounts of the active caspase (CASP)-3, CASP-6, CASP-7, and poly (ADP-ribose) polymerase-1 (PARP1, cleaved form), as well as DNA fragmentation in layilin-KD cells under apoptotic conditions induced by STS, using western blot and the DNA ladder method, respectively. Under STS-treated conditions, the amounts of active CASP-3, CASP-7, and poly (ADP-ribose) PARP1 were significantly smaller in layilin-KD cells than in control cells. Accordingly, DNA fragmentation was significantly suppressed in layilin-KD cells compared to control cells under STS-treated conditions., Conclusion: This study demonstrates that layilin contributes to ΔΨm reduction to promote apoptosis by up-regulating BAD and down-regulating BCL-2 in glioma cells. Our data elucidates a new function of layilin: regulation of mitochondria-mediated apoptosis., (© 2024. The Author(s).)

The most common mutation in southern Chinese individuals with late-onset multiple acyl-coenzyme A dehydrogenase deficiency (MADD; a fatty acid metabolism disorder) is c.250G > A (p.Ala84Thr) in the electron transfer flavoprotein dehydrogenase gene (ETFDH). Various phenotypes, including episodic weakness or rhabdomyolysis, exercise intolerance, and peripheral neuropathy, have been reported in both muscular and neuronal contexts. Our cellular models of MADD exhibit neurite growth defects and excessive apoptosis. Given that axonal degeneration and neuronal apoptosis may be regulated by B-cell lymphoma (BCL)-2 family proteins and mitochondrial outer membrane permeabilization through the activation of proapoptotic molecules, we measured the expression levels of proapoptotic BCL-2 family proteins (e.g., BCL-2-associated X protein and p53-upregulated modulator of apoptosis), cytochrome c, caspase-3, and caspase-9 in NSC-34 cells carrying the most common ETFDH mutation. The levels of these proteins were higher in the mutant cells than in the wide-type cells. Subsequent treatment of the mutant cells with coenzyme Q10 downregulated activated protein expression and mitigated neurite growth defects. These results suggest that the activation of the BCL-2/mitochondrial outer membrane permeabilization/apoptosis pathway promotes apoptosis in cellular models of MADD and that coenzyme Q10 can reverse this effect. Our findings aid the development of novel therapeutic strategies for reducing axonal degeneration and neuronal apoptosis in MADD., (© 2024. The Author(s).)

Objectives: To observe the protective effect of acupuncture on ovarian function and its impact on key molecules (Yes associated protein ［YAP］ and transcriptional coactivator with PDZ binding motif ［TAZ］) in the Hippo signaling pathway in poor ovarian response (POR) mice, in order to explore its mechanisms underlying improvement of POR by inhibiting ovarian cell apoptosis., Methods: The mice with regular motility cycle after screening were randomly divided into normal control, model and acupuncture groups, with 12 mice in each group. The POR model was established by gavage of tripterygium wilfordii polyglycoside suspension (50 mg · kg -1 ·d -1 ) for 2 weeks, and the mice of the normal control group was received an equal volume of 0.9% sodium chloride solution by gavage. The mice in the acupuncture group received manual acupuncture stimulation of bilateral "Taichong" (LR3) and "Sanyinjiao" (SP6), and "Zhongji" (CV3) and "Guanyuan" (CV4), with the filiform needles retained for 20 min, once daily for successive 10 days. The estrous cycle was determined by using vaginal exfoliated cell smear, and the body mass was detected weekly. The levels of serum anti Mullerian hormone (AMH), follicle stimulating hormone (FSH), estradiol (E2) and luteinizing hormone (LH) were measured using enzyme-linked immunosorbent assay (ELISA). Histopathological changes of the ovarian tissue were observed after H.E. staining, and the apoptosis of ovarian granulosa cells was measured using terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay. The expression levels of YAP, TAZ, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X (Bax), cysteine aspartic acid specific protease-3 (Caspase-3) mRNAs and proteins were detected using real-time PCR and Western blot, respectively., Results: Compared with the normal control group, the model group had an increase in the rate of estrous cycle disorder, estrous cycle, serum FSH and LH content, and apoptosis of ovarian granulosa cells, and expression levels of Bax and Caspase-3 mRNAs and p-YAP, Bax and Caspase-3 proteins ( P <0.01), and a decrease in the body mass, number of retrieved oocytes, ovarian wet weight and ovarian index, serum AMH and E2 contents, and the expression levels of YAP, TAZ, Bcl-2 mRNAs and proteins ( P <0.01). After acupuncture intervention, modeling induced increase and decrease of indexes mentioned above were completely reversed ( P <0.05, P <0.01). H.E. staining showed deformed ovarian structure, reduced number of normal developing follicles and increased number of atretic follicles, disordered arrangement of the granulosa cells with fewer hierarchy in the model group, which was improved in the acupuncture group, such as increase in the number and improvement in the shape of normal ovarian follicles and reduction of the atretic follicles., Conclusions: Acupuncture intervention can inhibit the apoptosis of ovarian granulosa cells and improve the ovarian function of POR mice, which may be related to its effects in up-regulating the expressions of YAP, TAZ (key molecules of Hippo signaling pathway).

BCL-w is a BCL-2 family protein that promotes cell survival in tissue- and disease-specific contexts. The canonical anti-apoptotic functionality of BCL-w is mediated by a surface groove that traps the BCL-2 homology 3 (BH3) α-helices of pro-apoptotic members, blocking cell death. A distinct N-terminal portion of BCL-w, termed the BCL-2 homology 4 (BH4) domain, selectively protects axons from paclitaxel-induced degeneration by modulating IP3 receptors, a noncanonical BCL-2 family target. Given the potential of BCL-w BH4 mimetics to prevent or mitigate chemotherapy-induced peripheral neuropathy, we sought to characterize the interaction between BCL-w BH4 and the IP3 receptor, combining "staple" and alanine scanning approaches with molecular dynamics simulations. We generated and identified stapled BCL-w BH4 peptides with optimized IP3 receptor binding and neuroprotective activities. Point mutagenesis further revealed the sequence determinants for BCL-w BH4 specificity, providing a blueprint for therapeutic targeting of IP3 receptors to achieve neuroprotection., Competing Interests: Declaration of interests M.F.P.-M., G.H.B., R.A.S., and L.D.W. are named co-inventors on international patent application WO 2018/039545 (and associated patent applications and granted national patents) related to this work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aim: To characterize the immunohistochemical expression of MDM2, Bcl-2, SATB2 and Ki-67 in histological variants of unicystic ameloblastoma (UA)., Methodology: Following the ethical approval, forty (40) patients with unicystic ameloblastoma were retrieved from the archives and subjected to immunohistochemistry (IHC). Sociodemographic and clinical data were also retrieved. The results were entered into a Microsoft Excel spreadsheet and analyzed using SPSS software., Results: Human tooth germs, which served as the control, showed moderate expression of Bcl-2 and MDM2 with slight proliferative activity in ameloblasts and moderate expression of SATB2 in ectomesenchyme and odontoblasts. Luminal UA (Type 1) showed low Ki-67 index and negative to mild Bcl-2 and MDM2 expression, whilst Type 1.2 (luminal and intraluminal), Type 1.2.3 (luminal, intraluminal and mural), and Type 1.3 (luminal and mural), including the recurrent cases, showed moderate to intense expression with high mean Ki-67 index. The difference between the study groups was statistically significant (p value < 0.001). No expression of SATB2 was noted in any histological variant of UA. Furthermore, no significant differences were noted in age, gender, site and location between the groups., Conclusion: In contrast to luminal variant of UA, mural±intraluminal variants and recurrent cases demonstrate higher expression of Bcl-2 and MDM2 with higher mean Ki-67 index. It may thus be prudent to provide aggressive treatment for cases, not just with mural follicles but also for the patients with intraluminal plexiform proliferation, to prevent recurrence and improve patient outcomes., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

We demonstrate the role of signaling via the glucocorticoid receptor, NR3C1, in differentiation of CD8+ T cell memory. Pharmacological inhibition as well as the short hairpin RNA-mediated knockdown of the receptor hindered memory transition and limited the homeostatic turnover of the activated CD8+ T cells. Dexamethasone exposure of CD8+ T cells expanded during a resolving infection with influenza A virus or a γ-herpesvirus promoted conversion of effector cells into memory cells by modulating cellular metabolism and lowering the accumulation of reactive oxygen species. Reduced reactive oxygen species levels in the responding effector cells upregulated Bcl2 and enhanced survival. The generated virus-specific memory CD8+ T cells were efficiently recalled following challenge of animals with a secondary infection to control it better. The memory-enhancing effect was predominantly evident at low doses of dexamethasone. Therefore, controlled glucocorticoid signaling within the effector CD8+ T cells is crucial for optimal memory differentiation., (Copyright © 2024 by The American Association of Immunologists, Inc.)

In Caenorhabditis elegans , apoptosis is inhibited by the BCL-2 homolog CED-9. Although canonically anti-apoptotic, CED-9 has a poorly understood pro-apoptotic function. CED-9 is thought to inhibit apoptosis by binding to and inhibiting the pro-apoptotic C. elegans APAF-1 homolog CED-4. We show that CED-9 or CED-4 mutations located in their CED-9-CED-4 binding regions reduce apoptosis without affecting the CED-9 anti-apoptotic function. These mutant CED-9 and CED-4 proteins are defective in a CED-9-CED-4 interaction in vitro and in vivo, revealing that the known CED-9-CED-4 interaction is required for the pro-apoptotic but not for the anti-apoptotic function of CED-9. The pro-apoptotic CED-9-CED-4 interaction occurs at mitochondria. In mammals, BCL-2 family members can activate APAF-1 via cytochrome c release from mitochondria. The conserved role of mitochondria in CED-9/BCL-2-dependent CED-4/APAF-1 activation is notable and suggests that understanding how CED-9 promotes apoptosis in C. elegans could inform the understanding of mammalian apoptosis and how disruptions of apoptosis promote certain human disorders.

Introduction: Pituitary adenomas (PAs) are benign tumors with high prevalence and, occasionally, aggressive course. The tumorigenesis of these lesions is not completely understood at the molecular level. BAK1 and BAX proteins play fundamental roles in apoptosis and seem to interact with VDAC proteins, whose expressions have been markedly altered in cancer, impacting their prognosis., Objective: to evaluate the gene expression of VDAC1, VDAC2, BAK1 and BAX and their association with clinical and imaging characteristics in PA., Methods: Clinical-epidemiological data were collected from 117 tumor samples from patients affected by PA. Invasiveness was assessed by the Knosp scale. Gene expression was examined by real-time PCR. Relative expression analysis was performed by 2^(-DDCt) method., Results: The sample was mainly composed of women (69/117 - 57.2%). Tumor subtypes observed were Non-Functioning (NF) (73/117 - 62.4%), Acromegaly (24/117 - 20.5%) and Cushing's Disease (CD) (20/117 - 17.1%). Compared to normal tissue, there was a significant reduction in VDAC1 expression in the Acromegaly (p=0.029) and NF (p=0.002) groups. BAX expression was lower in all groups (p <0.001; p=0.007; P =0.005). No difference was found in VDAC2 and BAK1 expression, compared to normal pituitary. Overexpression of VDAC2 occurred in PAs with post-surgical regrowth (p=0.042). A strongly negative correlation was observed in BAX and BAK1 expression in CD., Conclusion: The results indicated that downregulations of VDAC1 and BAX may be related to resistance to apoptosis. In contrast, overexpression of VDAC2 in regrowing PAs suggests an antiapoptotic role for this gene. In summary, the genes evaluated might be involved in the biopathology of PAs., 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 © 2024 Facundo, Magalhães, Nascimento, Azulay, Santos, Freitas, Nascimento, Rodrigues, Santos, Beckman, Abreu, Silva, Carneiro, Oliveira Neto, Gil da Costa, Corcoy, Mato and Faria.)

Apoptosis is the main form of regulated cell death in metazoans. Apoptotic pathways are well characterized in nematodes, flies, and mammals, leading to a vision of the conservation of apoptotic pathways in metazoans. However, we recently showed that intrinsic apoptosis is in fact divergent among metazoans. In addition, extrinsic apoptosis is poorly studied in non-mammalian animals, making its evolution unclear. Consequently, our understanding of apoptotic signaling pathways evolution is a black box which must be illuminated by extending research to new biological systems. Lophotrochozoans are a major clade of metazoans which, despite their considerable biological diversity and key phylogenetic position as sister group of ecdysozoans (i.e. flies and nematodes), are poorly explored, especially regarding apoptosis mechanisms. Traditionally, each apoptotic signaling pathway was considered to rely on a specific initiator caspase, associated with an activator. To shed light on apoptosis evolution in animals, we explored the evolutionary history of initiator caspases, caspase activators, and the BCL-2 family (which control mitochondrial apoptotic pathway) in lophotrochozoans using phylogenetic analysis and protein interaction predictions. We discovered a diversification of initiator caspases in molluscs, annelids, and brachiopods, and the loss of key extrinsic apoptosis components in platyhelminths, along with the emergence of a clade-specific caspase with an ankyrin pro-domain. Taken together, our data show a specific history of apoptotic actors' evolution in lophotrochozoans, further demonstrating the appearance of distinct apoptotic signaling pathways during metazoan evolution., Competing Interests: Conflict of Interest The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The translocation t(14;18) activates BCL2 and is considered the initiating genetic lesion in most follicular lymphomas (FL). Surprisingly, FL patients fail to respond to the BCL2 inhibitor, Venetoclax. We show that mutations and deletions affecting the histone lysine methyltransferase SETD1B (KMT2G) occur in 7% of FLs and 16% of diffuse large B cell lymphomas (DLBCL). Deficiency in SETD1B confers striking resistance to Venetoclax and an experimental MCL-1 inhibitor. SETD1B also acts as a tumor suppressor and cooperates with the loss of KMT2D in lymphoma development in vivo. Consistently, loss of SETD1B in human lymphomas typically coincides with loss of KMT2D. Mechanistically, SETD1B is required for the expression of several proapoptotic BCL2 family proteins. Conversely, inhibitors of the KDM5 histone H3K4 demethylases restore BIM and BIK expression and synergize with Venetoclax in SETD1B-deficient lymphomas. These results establish SETD1B as an epigenetic regulator of cell death and reveal a pharmacological strategy to augment Venetoclax sensitivity in lymphoma., (© 2024 Portelinha et al.)

Backgrounds: Renal tubular injury caused by oxidative stress and inflammation results in acute kidney injury. Recent research reported that antibiotics may protect renal tubules from progressive deterioration, but the underlying mechanism remains unclear. Therefore, we investigated the efficacy and mechanism of action of antibiotics against renal tubular injury., Methods: We screened ciprofloxacin, ceftizoxime, minocycline, and netilmicin and selected ciprofloxacin to examine further because of its low toxicity towards renal tubular cells. We evaluated the effect of ciprofloxacin on cell survival by analyzing apoptosis and autophagy., Results: Terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL) assay results showed that the ciprofloxacin group had less apoptotic cells than the control group. The ratio of cleaved caspase 3 to caspase 3, the final effector in the apoptosis process, was decreased, but the ratio of B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) to Bcl-2 located upstream of caspase 3 was not decreased in the ciprofloxacin group. Therefore, apoptosis inhibition does not occur via Bax/Bcl-2. Conversely, the levels of phosphorylated Bcl-2, and Beclin-1, an autophagy marker, were increased, and that of caspase-3 was decreased in the ciprofloxacin group., Conclusion: This indicates that ciprofloxacin enhances autophagy, increasing the amount of free Beclin-1 via phosphorylated Bcl-2, and inhibits caspase activity. Therefore, ciprofloxacin might protect against renal tubular injury through the activation of autophagy in the setting of acute kidney injury., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Breast cancer is the predominant form of cancer among women and ranks as the second most prevalent cancer globally, affecting both developed and less developed countries. Presently, accessible cancer treatment methods either employ recently created, secure, and efficient chemotherapeutic medications or directly target innovative pathways that cause apoptosis. One of the indirect strategies for treating this fatal illness has mostly depended on its essential role in cell cycle arrest and apoptosis induction, as well as the antagonistic interaction between the Bcl-2 and Mcl-1 proteins, in order to avert major health repercussions. We reported that newly synthesized fused chromenopyrimidines ( 3a and 4a ) showed potential cell cycle arrest and dual Bcl-2 and Mcl-1 inhibitory characteristics. Bcl-2 and Mcl-1 were the targets of a molecular docking procedure. The previous docking results are in line with the biological data and suggest that 3a may have promising anti-cancer activity.

Cutaneous squamous cell carcinoma (cSCC) is a common form of skin cancer, but treatments for advanced cases have limited efficacy. Trophoblast cell-surface antigen 2 (TROP2) is a cell-surface protein that is widely expressed in various tumours, where it exerts significant influence over critical processes such as tumour cell growth, apoptosis, migration, invasion and metastasis. Sacituzumab govitecan, an antibody-drug conjugate (ADC) targeting TROP2, is emerging as a promising strategy for anticancer therapy. In this study, we investigated TROP2 expression in cSCC tissues from 51 patients and evaluated its function in the A431 human SCC cell line. Immunohistochemical analysis revealed TROP2 expression on the plasma membrane of cSCC tissues and A431 cells. A431 cells showed sensitivity to sacituzumab govitecan with a significant concentration-dependent decrease in viable cell number. In addition, Knockdown of TROP2 resulted in decreased expression of cyclin D1 and BCL-2, along with reduced cell viability. Knockdown of TROP2 also resulted in decreased expression of vimentin, along with reduced migratory capacity. These findings suggest that TROP2 plays a crucial role in cSCC cell proliferation and migration, and highlight the potential of sacituzumab govitecan as a promising therapeutic option for cSCC., (© 2024 The Author(s). Experimental Dermatology published by John Wiley & Sons Ltd.)

Arsenic is a toxic environmental pollutant heavy metal, and one of its critical target tissues in the body is the liver. Carvacrol is a natural phytocompound that stands out with its antioxidant, anti-inflammatory, and antiapoptotic properties. The current study aims to investigate the protective feature of carvacrol against sodium arsenite-induced liver toxicity. Thirty-five Sprague-Dawley male rats were divided into five groups: Control, Sodium arsenite (SA), CRV, SA + CRV25, and SA + CRV50. Sodium arsenite was administered via oral gavage at a dose of 10 mg/kg for 14 days, and 30 min later, CRV 25 or 50 mg/kg was administered via oral gavage. Oxidative stress, inflammation, apoptosis, autophagy damage pathways parameters, and liver tissue integrity were analyzed using biochemical, molecular, western blot, histological, and immunohistological methods. Carvacrol decreased sodium arsenite-induced oxidative stress by suppressing malondialdehyde levels and increasing superoxide dismutase, catalase, glutathione peroxidase activities, and glutathione levels. Carvacrol reduced inflammation damage by reducing sodium arsenite-induced increased levels of NF-κB and the cytokines (TNF-α, IL-1β, IL-6, RAGE, and NLRP3) it stimulates. Carvacrol also reduced sodium arsenite-induced autophagic (Beclin-1, LC3A, and LC3B) and apoptotic (P53, Apaf-1, Casp-3, Casp-6, Casp-9, and Bax) parameters. Carvacrol preserved sodium arsenite-induced impaired liver tissue structure. Carvacrol alleviated toxic damage by reducing sodium arsenite-induced increases in oxidative stress, inflammation, apoptosis, and autophagic damage parameters in rat liver tissues. Carvacrol was also beneficial in preserving liver tissue integrity., (© 2024 The Author(s). Journal of Biochemical and Molecular Toxicology published by Wiley Periodicals LLC.)

The plausible effects of SARS-CoV-2 infection on the expression of anti/proapoptotic molecules have been suspected. This cohort study examined the expression of p53, Bcl-2, Bid, Bak, and Bax molecules, the genes associated with induction or inhibition of apoptosis, in the SARS-CoV-2-infected patients with severe and mild symptoms in an Iranian population. In this 6-month cohort study, the expression of p53, Bcl-2, Bid, Bak, and Bax molecules was evaluated at onset of diagnosis, 24 h after symptom onset, and 6 months later in the nasopharyngeal cells of SARS-CoV-2-infected hospitalized patients and outpatients in comparison with healthy controls using the real-time PCR technique. At the onset of the study, the relative expression of p53, Bcl-2, Bid, Bak, and Bax significantly increased in the SARS-CoV-2-infected hospitalized patients and decreased after 6 months. The healthy controls showed potential positive correlations among the molecules, but the patients did not show these correlations. Since SARS-CoV-2 needs host cell survival, it appears that the virus induces the expression of Bcl-2 as an antiapoptotic molecule, and the host cells upregulate the proapoptotic molecules to neutralize the effects. Dysregulation of correlation expression of the molecules among the patients proved that SARS-CoV-2 affects the expression of the molecules involved in apoptosis. SARS-CoV-2 could be considered an important factor that regulates the expression of several molecules participating in cancer pathogenesis.

Introduction: The combined use of the BCL-2 inhibitor venetoclax with azacitidine now is the standard of care for patients with acute myeloid leukemia (AML) unfit for intensive chemotherapy with outcomes exceeding those achieved with hypomethylating agents alone. Venetoclax in combination with intensive chemotherapy is also increasingly used both as frontline as well as salvage therapy. However, resistance to and relapse after venetoclax-based therapies are of major concern and outcomes after treatment failure remain poor., Areas Covered: A comprehensive search was performed using PubMed database (up to April 2024). Studies evaluating venetoclax-based combination treatments in AML and studies assessing markers of response and resistance to venetoclax were investigated. We summarize the status of venetoclax-based therapies in the frontline and relapsed/refractory setting with focus on the main mechanisms of resistance to BCL-2 inhibition. Further, strategies to overcome resistance including combinatorial regimens of hypomethylating agent (HMA) + venetoclax + inhibitors targeting actionable mutations like IDH1/2 or FLT3-ITD and the introduction of novel agents like menin-inhibitors are addressed., Expert Opinion: Although venetoclax is reshaping the treatment of unfit and fit AML patients, prognosis of patients after HMA/VEN failure remains dismal, and strategies to abrogate primary and secondary resistance are an unmet clinical need.

Lung cancer is one of the most common malignant tumors. Despite decades of research, the treatment of lung cancer remains challenging. Non-small cell lung cancer (NSCLC) is the primary type of lung cancer and is a significant focus of research in lung cancer treatment. The deubiquitinase ubiquitin-specific protease 28 (USP28) plays a role in the progression of various tumors and serves as a potential therapeutic target. This study aims to determine the role of USP28 in the progression of NSCLC. We examined the impact of the USP28 inhibitor AZ1 on the cell cycle, apoptosis, DNA damage response, and cellular immunogenicity in non-small cell lung cancer. We observed that AZ1 and siUSP28 induce DNA damage, leading to the activation of Noxa-mediated mitochondrial apoptosis. The dsDNA and mtDNA released from DNA damage and mitochondrial apoptosis activate tumor cell immunogenicity through the cGAS-STING signaling pathway. Simultaneously, targeting USP28 promotes the degradation of c-MYC, resulting in cell cycle arrest and inhibition of DNA repair. This further promotes DNA damage-induced cell apoptosis mediated by the Noxa protein, thereby enhancing tumor cell immunogenicity mediated by dsDNA and mtDNA. Moreover, we found that the combination of AZ1 and cisplatin (DDP) can enhance therapeutic efficacy, thereby providing a new strategy to overcome cisplatin resistance in NSCLC. These findings suggest that targeting USP28 and combining it with cisplatin are feasible strategies for treating NSCLC., (© 2024. The Author(s).)

Objective: To analyze the immunohistochemical expression of YAP and its correlation with markers involved in cell proliferation and apoptosis in benign epithelial odontogenic lesions., Study Design: The sample consisted of 95 cases of odontogenic lesions (25 dentigerous cysts, 30 non-syndromic odontogenic keratocysts, 30 conventional ameloblastomas, and 10 unicystic ameloblastomas) and 10 dental follicles used as normal odontogenic tissue. The histological sections were submitted to immunohistochemistry with YAP, cyclin D1, Ki-67, and Bcl-2 antibodies. Immunoexpression was analyzed qualitatively and quantitatively using an adapted method. The collected data were analyzed descriptively and statistically (p ≤ 0.05)., Results: The highest YAP expression was observed in odontogenic keratocysts, followed by unicystic ameloblastomas and conventional ameloblastomas, which exhibited moderate immunoreactivity predominantly in peripheral cells. Furthermore, significant differences in YAP immunoexpression were observed between the groups analyzed, with significant positive correlations between YAP and cyclin D1 in dentigerous cysts and unicystic ameloblastomas and between YAP and Ki-67 in unicystic ameloblastomas (p < 0.05). However, there were no statistically significant correlations between YAP and Bcl-2 immunoexpression in the groups studied., Conclusion: YAP may influence epithelial cell proliferation in odontogenic cysts and tumors, suggesting its possible participation in the progression of the odontogenic lesions studied., (© 2024 Wiley Periodicals LLC.)

Appropriate biomarkers are required to predict the clinical outcome of triple-negative breast cancer (TNBC). In this study, we focused on the clinical importance of two representative tumor-associated proteins, Bcl-2 and p53. Bcl-2 expression is usually related to estrogen receptor expression and a favorable outcome in breast cancer. TNBC has been reported to show a high frequency of p53 positivity suggesting TP53 mutations. The expressions of Bcl-2 and p53 were immunohistochemically examined in TNBC involving two age groups of postmenopausal women (≥75 y/o, n = 75; 55-64 y/o, n = 47), who underwent surgery without neoadjuvant therapy. We examined their associations with each other, or with clinicopathological factors including the outcome. Bcl-2 expression was inversely correlated with androgen receptor, apocrine morphology, and p53 expressions, and was an independent predictor of a poor outcome in total or in younger women. p53 positivity was associated with a more favorable outcome than p53 negativity in the younger group. In combined analyzes, none of the twenty Bcl-2-negative/p53-positive cases in the younger group exhibited recurrence, resulting in the independent favorable predictive value of Bcl-2-negative/p53-positive. The anti-apoptotic nature of Bcl-2 may be apparent in TNBC. The excellent outcome of Bcl-2-negative/p53-positive cases in the younger group warrants further combined investigation of Bcl-2/p53 in TNBC., (© 2024 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Here, we revealed the reversibility of cabazitaxel (CBZ)-induced apoptosis in PC-3 castration resistant metastatic prostate cancer cells (mCRPC) through the hallmarks of apoptosis. The recovery of PC-3 cells from apoptosis upon removal of CBZ at different recovery periods was evaluated by Annexin V, DNA damage, oxidative damage, mitochondrial membrane depolarization, and caspase activation. Our results showed that the administration of CBZ caused apoptosis for 72 h in PC-3 cells. However, recovered cells exhibited decreased nuclear damage, plasma membrane disruption, ROS level, release cytochrome c level and caspase-3 activation with upregulation of Bcl-2 expression upon removal of especially 1 nM CBZ for 24 h recovery period in PC-3 cells. Our study indicates that CBZ treated PC-3 cells can recover after apoptotic cell death. However, advanced molecular analysis should elucidate the relationship between the molecular mechanisms of recovery and taxane response or resistance in PC-3 mCRPC cells., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Increasing the levels of antiapoptotic Bcl-2 proteins is an important way that cancer cells utilize to get out of apoptosis, underscoring their significance as promising targets for anticancer therapies. Lately, a primary compound 1 bearing thiazolidine-2,4-dione was discovered to exhibit comparable Mcl-1 inhibitory activity in comparison to WL-276. Herein, thirty-nine thiazolidine-2,4-dione analogs were yielded through incorporating different biphenyl moieties (R 1 ), amino acid side chains (R 2 ) and sulfonamides (R 3 ) on 1. The findings indicated that certain compounds exhibited favorable inhibitory effects against Bcl-2/Mcl-1, while demonstrating limited or negligible binding affinity towards Bcl-xL. In particular, compounds 16 and 20 exhibited greater Bcl-2/Mcl-1 inhibition compared to AT-101, WL-276 and 1. Moreover, they demonstrated notable antiproliferative effects and significantly induced apoptosis in U937 cells. The western blot and co-immunoprecipitation assays confirmed that 20 could induce alterations in the expression of apoptosis-associated proteins to result in apoptosis through on-target Bcl-2 and Mcl-1 inhibition. In addition, 20 exhibited favorable stability profiles in both rat plasma and rat liver microsomes. In total, 20 could be used as a promising compound to discover Bcl-2/Mcl-1 dual inhibitors with favorable therapeutic properties., 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 Inc. All rights reserved.)

We present a series of 9 follicular lymphomas that progressed/transformed into classical Hodgkin lymphoma (CHL). Three cases of CHL showed a syncytial pattern (SCHL) making the differential diagnosis to Gray zone lymphoma (GZL) challenging. None of these three cases presented in the mediastinum. Based in all molecular data analyzed (BCL2/BCL6 FISH studies, IgH PCR and TNGS with a customized gene panel) we did find clonal relationship between the BCL2-positive FL cases and their CHL components in all cases. The three SCHL/GZL cases showed an activated phenotype according to Hans algorithm, presented the t(14; 18)(q32; q21), two out of three showed B cell markers and all expressed CD30 and p53. Interestingly, we identified three BCL2-negative FL cases with a further diagnosis of CHL expanding the spectrum of these association. In one of these three cases a different mutational profile was found in both the FL and the CHL components. All this data together suggests that CHL associated to BCL2-positive FL could be originated in a common progenitor cell (CPC) that give rise to both FL and CHL, acquiring this last component further genetic events in a linear fashion. On the other hand, no clonal relationship between CHL and BCL2-negative FL could be found, suggesting a fortuity association. Nevertheless, ample series of cases studied with more sensitive techniques are needed to confirm our hypothesis., Competing Interests: Declaration of competing interest None of the authors have any relevant conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Hypothesis: Memantine, an N -methyl- d -aspartate receptor antagonist, is widely used to treat Alzheimer's disease and has been found to have potential neuroprotective effects. In this study, we evaluated the protective effects of memantine against cisplatin-induced ototoxicity., Background: Cisplatin is a widely used anticancer drug for various cancers; however, its use is limited by its side effects, including ototoxicity. Several drugs have been developed to reduce cisplatin toxicity. In this study, we treated cisplatin-damaged cochlear hair cells with memantine and evaluated its protective effects., Method: House Ear Institute Organ of Corti 1 (HEI-OC1) cells and cochlear explants were treated with cisplatin or memantine. Cell viability, apoptotic patterns, reactive oxygen species (ROS) production, Bcl-2/caspase-3 activity, and cell numbers were measured to evaluate the anti-apoptotic and antioxidative effects of memantine., Result: Memantine treatment significantly improved cell viability and reduced cisplatin-induced apoptosis in auditory cells. Bcl-2/caspase-3 activity was also significantly increased, suggesting anti-apoptotic effects against cisplatin-induced ototoxicity., Conclusion: Our results suggest that memantine protects against cisplatin-induced ototoxicity in vitro, providing a potential new strategy for preventing hearing loss in patients undergoing cisplatin chemotherapy., Competing Interests: The authors disclose no conflicts of interest., (Copyright © 2024, Otology & Neurotology, Inc.)

MicroRNAs (miRNAs) are a class of small non-coding RNAs that act as important regulators of gene expression, involved in various biological pathways. Aberrant miRNAs expression is associated with the onset and progression of colorectal cancer (CRC). The aim of this study was to investigate the correlation between five miRNAs (miR-29a, miR-101, miR-125b, miR-146a, and miR-155), found to be deregulated in tissue samples of CRC patients, and clinicopathological characteristics and histological markers. Analysis of histological markers was performed by immunohistochemical staining of tumour tissues with Ki-67, p53, CD34, and Bcl-2. Our findings revealed a significant negative correlation between miR-29a expression and Bcl-2 levels. Furthermore, high miR-29a expression was associated with a lower incidence of distant metastasis in CRC patients. We observed negative correlations between miR-101 expression and the number of lymph nodes with metastasis, as well as the size of the largest metastasis; miR-125b expression and lymphovascular invasion; and miR-155 expression and mucus presence. Our survival analysis demonstrated that high miR-29a expression correlated with better progression-free survival of CRC patients, underscoring its potential as a prognostic marker. Our study unveiled intricate relationships between specific miRNA expressions and clinicopathological features in CRC, highlighting the potential utility of miR-29a as a valuable prognostic biomarker., 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 GmbH. All rights reserved.)

Background: Inflammation and oxidative stress are key players in lung injury stemming from cardiac ischemia (LISCI). Cannabidiol (CBD) demonstrates tissue-protective properties through its antioxidant, anti-inflammatory, and anti-apoptotic characteristics. This study aims to assess the preventive (p-CBD) and therapeutic (t-CBD) effects of CBD on LISCI., Methods: Forty male Wistar Albino rats were divided into four groups: control (CON), LISCI, p-CBD, and t-CBD. The left anterior descending coronary artery was ligated for 30 min of ischemia followed by 30 min of reperfusion. Lung tissues were then extracted for histopathological, immunohistochemical, genetic, and biochemical analyses., Results: Histopathologically, marked hyperemia, increased septal tissue thickness, and inflammatory cell infiltrations were observed in the lung tissues of the LISCI group. Spectrophotometrically, total oxidant status and oxidative stress index levels were elevated, while total antioxidant status levels were decreased. Immunohistochemically, expressions of cyclooxygenase-1 (COX1), granulocyte colony-stimulating factor (GCSF), interleukin-6 (IL6) were increased. In genetic analyses, PERK and CHOP expressions were increased, whereas Nuclear factor erythroid 2-related factor 2 (NRF2) and B-cell leukemia/lymphoma 2 protein (BCL2) expressions were decreased. These parameters were alleviated by both prophylactic and therapeutic CBD treatment protocols., Conclusion: In LISCI-induced damage, both endoplasmic reticulum and mitochondrial stress, along with oxidative and inflammatory markers, were triggered, resulting in lung cell damage. However, both p-CBD and t-CBD treatments effectively reversed these mechanisms, normalizing all histopathological, biochemical, and PCR parameters.

Carbon monoxide (CO) is produced via incomplete combustion of fossil fuels and it may cause long-term neurological sequel upon exposure. Hesperidin (HES), a flavanone glycoside found in citrus plants, exerts diverse beneficial health effects. The present study mechanistically examined the neuroprotective effects of HES in CO-poisoned rats. Thirty male Wistar rats (five groups of six animals) were exposed to 3000 ppm CO for 1 h. Immediately after the exposure and on the next 4 consecutive days (totally five doses), rats intraperitoneally received either normal saline (the control group) or different doses of HES (25, 50, and 100 mg/kg). A sham group that was not exposed to CO was also considered. After evaluation of spatial learning and memory using a Morris water maze (MWM), animals were sacrificed and oxidative stress status in blood samples, and Akt, Bax, Bcl2, and brain-derived neurotrophic factor (BDNF) expression in brain samples were assessed. Western blot analysis indicated increased Akt but decreased Bax/Bcl2 levels in the HES 100 mg/kg, and induced BDNF levels in all HES-treated groups. MWM results showed that HES significantly decreased memory loss. The current findings indicate that HES could alleviate neurological impairments induced by CO in rats., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Cholangiocarcinoma (CCA) is a malignancy of the bile duct epithelium that is commonly found in the Thai population. CCA has poor prognosis and a low survival rate due to the lack of early diagnosis methods and the limited effectiveness of current treatments. A number of oncogenic spliced-transcripts resulting from mRNA splicing errors have been reported in CCA, and aberrant mRNA splicing is suspected to be a key driver of this cancer type. The hyperphosphorylation of serine/arginine rich-splicing factors (SRSFs) by serine/arginine protein kinases (SRPKs) causes them to translocate to the nucleus where they facilitate gene splicing errors that generate cancer-related mRNA/protein isoforms., Methods: The correlation between SRPK expression and the survival of CCA patients was analyzed using data from The Cancer Genome Atlas (TCGA) dataset. The effect of SRPK inhibitors (SRPIN340 and SPHINX31) on two CCA cell lines (KKU-213A and TFK-1) was also investigated. The induction of cell death was studied by Calcein-AM/PI staining, AnnexinV/7AAD staining, immunofluorescence (IF), and Western blotting (WB). The phosphorylation and nuclear translocation of SRSFs was tracked by WB and IF, and the repair of splicing errors was examined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR)., Results: High levels of SRPK1 and SRPK2 transcripts, and in particular SRPK1, correlated with shorter survival in CCA patients. SRPIN340 and SPHINX31 increased the number of dead and apoptotic cells in a dose-dependent manner. CCA also showed diffuse expression of cytoplasmic cytochrome C and upregulation of cleaved caspase-3. Moreover, SRSFs showed low levels of phosphorylation, resulting in the accumulation of cytoplasmic SRSF1. To link these phenotypes with aberrant gene splicing, the apoptosis-associated genes Bridging Integrator 1 ( BIN1 ), Myeloid cell leukemia factor 1 ( MCL-1 ) and B-cell lymphoma 2 ( BCL2 ) were selected for further investigation. Treatment with SRPIN340 and SPHINX31 decreased anti-apoptotic BIN1+12A and increased pro-apoptotic MCL-1S and BCL-xS ., Conclusions: The SRPK inhibitors SRPIN340 and SPHINX31 can suppress the phosphorylation of SRSFs and their nuclear translocation, thereby producing BIN1 , MCL-1 and BCL2 isoforms that favor apoptosis and facilitate CCA cell death., (© 2024 The Author(s). Published by IMR Press.)

Background: Acute myeloid leukaemia (AML) comprises a group of heterogeneous and aggressive haematological malignancies with unsatisfactory prognoses and limited treatment options. Treatments targeting B-cell lymphoma-2 (BCL-2) with venetoclax have been approved for patients with AML, and venetoclax-based drug combinations are becoming the standard of care for older patients unfit for intensive chemotherapy. However, the therapeutic duration of either single or combination strategies is limited, and the development of resistance seems inevitable. Therefore, more effective combination regimens are urgently needed., Methods: The efficacy of combination therapy with NL101, a SAHA-bendamustine hybrid, and venetoclax was evaluated in preclinical models of AML including established cell lines, primary blasts from patients, and animal models. RNA-sequencing and immunoblotting were used to explore the underlying mechanism., Results: NL101 significantly potentiated the activity of venetoclax in AML cell lines, as evidenced by the enhanced decrease in viability and induction of apoptosis. Mechanistically, the addition of NL101 to venetoclax decreased the stability of the antiapoptotic protein myeloid cell leukaemia-1 (MCL-1) by inhibiting ERK, thereby facilitating the release of BIM and triggering mitochondrial apoptosis. Moreover, the strong synergy between NL101 and venetoclax also relied on the downregulation of c-Myc via PI3K/Akt/GSK3β signalling. The combination of NL101 and venetoclax synergistically eliminated primary blasts from 10 AML patients and reduced the leukaemia burden in an MV4-11 cell-derived xenograft model., Conclusions: Our results encourage the pursuit of clinical trials of combined treatment with NL101 and venetoclax and provide a novel venetoclax-incorporating therapeutic strategy for AML., (© 2024. The Author(s).)

LL-37 can inhibit the growth of K562 cancer cells when it is conjugated with iron oxide nanoparticles. In this study, Fe3O4 nanoparticles were synthesized using the co-precipitation method and then modified with the LL-37 peptide through an NH2 bridge. The accuracy of the synthesis process was confirmed through various analytical tests, including FTIR, XRD, FESEM, and EDX. To assess the treatment's effectiveness, a viability test was carried out on K562 leukemia cells and normal peripheral blood mononuclear cells. In addition, flow cytometry and Hoechst staining were used to investigate the mechanism of action of the drug. The expression levels of the Bcl-2, Bax, and TP53 genes in the treated cells and the control group were measured using qRT-PCR. The results indicated that the size of the nanoparticles ranged between 34 and 40 nm. The NH2@LL-37@Fe3O4 nanoparticles more effectively inhibited the growth of cancer cells in a concentration-dependent manner, as compared to Fe3O4 alone. Further analysis revealed that apoptosis occurred through increased expression of TP53 and Bax genes compared to the Bcl-2 gene. Therefore, induction of apoptosis and inhibition of growth in K562 cells was attributed to the impact of iron oxide magnetic nanoparticles conjugated with the LL-37 peptide through the TP53/Bax/Bcl-2 pathway., (© 2024. The Author(s).)

Bfl-1, a member of the Bcl-2 family of proteins, plays a crucial role in apoptosis regulation and has been implicated in cancer cell survival and resistance to venetoclax therapy. Due to the unique cysteine residue in the BH3 binding site, the development of covalent inhibitors targeting Bfl-1 represents a promising strategy for cancer treatment. Herein, the optimization of a covalent cellular tool from a lead-like hit using structure based design is described. Informed by a reversible X-ray fragment screen, the strategy to establish interactions with a key glutamic acid residue (Glu78) and optimize binding in a cryptic pocket led to a 1000-fold improvement in biochemical potency without increasing reactivity of the warhead. Compound ( R,R,S )-26 has a k inact /K I of 4600 M -1 s -1 , shows <1 μM caspase activation in a cellular assay and cellular target engagement, and has good physicochemical properties and a promising in vivo profile.

Objective: To investigate the molecular mechanism of autophagy and apoptosis induced by ultrafine carbon black in human bronchial epithelial cells (BEAS-2B cells), and to study the intervention effect and mechanism of N-acetylcysteine (NAC) on ultrafine carbon black-induced oxidative damage in BEAS-2B cells. Methods: In March 2023, BEAS-2B cells were used as research object, an in vitro airway model exposed to ultrafine carbon black was constructed. A control group and three carbon black exposure groups (50, 100, 200 μg/ml) were set up, and the cells were treated with corresponding concentrations of ultrafine carbon black for 24 hours. In addition, the experiment was divided into control group, NAC+ control group, 100 μg/ml carbon black exposure group and NAC+ exposure group. The corresponding groups were treated with 2 mmol/L NAC for 1 h and 100 μg/ml ultrafine carbon black for 24 h, respectively. Cell viability was measured by CCK-8 assay. Intracellular reactive oxygen species (ROS) level was detected by chemical fluorescence method. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), as well as the content of malondialdehyde (MDA) were detected by colorimetry. The mRNA and protein expressions of autophagy-related genes[Atg5, Atg7, Beclin1, microtubule-associated protein light chain 3B (LC3B), p62 and lysosome-associated membrane protein 2 (LAMP2) ] and apoptosis-related genes [B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase3, Caspase9 and poly (ADP-ribose) polymerase 1 (PARP1) ] were determined by fluorescence quantitative PCR and Western blot. Cell apoptosis was determined by flow cytometry. Results: Compared with the control group, the relative survival rates of BEAS-2B cells in 50, 100, 200 μg/ml carbon black exposure groups were significantly decreased, the levels of ROS and MDA were significantly increased, and the activities of SOD, GSH-Px and CAT were significantly decreased ( P <0.05). The relative survival rate, ROS and MDA levels, SOD, GSH-Px and CAT activities were significantly correlated with the exposure dose of ultrafine carbon black ( r (s)=-0.755, 0.826, 0.934, -0.810, -0.880, -0.840, P <0.05). Compared with the control group, the relative expression levels of Atg5, Atg7, Beclin1, LC3B, p62, LAMP2, Bax, Caspase3, Caspase9, PARP1 mRNA and Atg5, Atg7, Beclin1, LC3BⅡ, p62, LAMP2, Bax, cleaved Caspase3 (C-Caspase3), cleaved Caspase9 (C-Caspase9), cleaved PARP1 (C-PARP1) protein and the ratio of LC3BⅡ/LC3BⅠ in 50, 100 and 200 μg/ml carbon black exposure groups were significantly increased, while the relative expression levels of Bcl-2 mRNA and protein were significantly decreased ( P <0.05). The changes of the above indexes were significantly correlated with the exposure dose of carbon black ( r (s)=0.892, 0.879, 0.944, 0.892, 0.828, 0.880, 0.814, 0.794, 0.931, 0.918, 0.813, 0.866, 0.774, 0.695, 0.918, 0.761, 0.794, 0.944, 0.833, 0.866, 0.905, -0.886, -0.748, P <0.05). Compared with 100 μg/ml carbon black exposure group, the relative survival rate, the activities of SOD, GSH-Px and CAT in NAC+exposure group were significantly increased, while the levels of ROS and MDA were significantly decreased, and the relative expression levels of LC3B, p62 and Caspase3 mRNA and protein as well as the ratio of LC3BⅡ/LC3BⅠ were significantly decreased, and the differences were statistically significant ( P <0.05). Compared with the control group, the apoptosis rates of BEAS-2B cells in 50, 100, 200 μg/ml carbon black exposure groups were significantly increased ( P <0.05), and there was a significant positive correlation between ultrafine carbon black exposure dose and cell apoptosis rate ( r (s)=0.944, P <0.05). While compared with 100 μg/ml carbon black exposure group, the apoptosis rate of NAC+exposure group was significantly decreased, and the difference was statistically significant ( P <0.05) . Conclusion: Cell autophagy and apoptosis may be important pathophysiological mechanisms of ultrafine carbon black-induced oxidative damage in BEAS-2B cells. NAC can alleviate the occurrence of BEAS-2B cell damage caused by ultrafine carbon black by regulating oxidative stress and the cascading autophagy and apoptosis pathways.

The widespread use of potent androgen receptor signaling inhibitors (ARSIs) has led to an increasing emergence of AR-independent castration-resistant prostate cancer (CRPC), typically driven by loss of AR expression, lineage plasticity, and transformation to prostate cancers (PCs) that exhibit phenotypes of neuroendocrine or basal-like cells. The anti-apoptotic protein BCL2 is upregulated in neuroendocrine cancers and may be a therapeutic target for this aggressive PC disease subset. There is an unmet clinical need, therefore, to clinically characterize BCL2 expression in metastatic CRPC (mCRPC), determine its association with AR expression, uncover its mechanisms of regulation, and evaluate BCL2 as a therapeutic target and/or biomarker with clinical utility. Here, using multiple PC biopsy cohorts and models, we demonstrate that BCL2 expression is enriched in AR-negative mCRPC, associating with shorter overall survival and resistance to ARSIs. Moreover, high BCL2 expression associates with lineage plasticity features and neuroendocrine marker positivity. We provide evidence that BCL2 expression is regulated by DNA methylation, associated with epithelial-mesenchymal transition, and increased by the neuronal transcription factor ASCL1. Finally, BCL2 inhibition had antitumor activity in some, but not all, BCL2-positive PC models, highlighting the need for combination strategies to enhance tumor cell apoptosis and enrich response.

Objective: Long-term exposure to arsenic has been linked to several illnesses, including hypertension, diabetes, hepatic and renal diseases and cardiovascular malfunction. The aim of the current investigation was to determine whether zingerone (ZN) could shield rats against the hepatotoxicity that sodium arsenite (SA) causes., Methods: The following five groups of thirty-five male Sprague Dawley rats were created: I) Control; received normal saline, II) ZN; received ZN, III) SA; received SA, IV) SA + ZN 25; received 10 mg/kg body weight SA + 25 mg/kg body weight ZN, and V) SA + ZN 50; received 10 mg/kg body weight SA + 50 mg/kg body weight ZN. The experiment lasted 14 days, and the rats were sacrificed on the 15th day. While oxidative stress parameters were studied by spectrophotometric method, apoptosis, inflammation and endoplasmic reticulum stress parameters were measured by RT-PCR method., Results: The SA disrupted the histological architecture and integrity of the liver and enhanced oxidative damage by lowering antioxidant enzyme activity, such as those of glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), glutathione (GSH) level and increasing malondialdehyde (MDA) level in the liver tissue. Additionally, SA increased the mRNA transcript levels of Bcl2 associated x (Bax), caspases (-3, -6, -9), apoptotic protease-activating factor 1 (Apaf-1), p53, tumor necrosis factor-α (TNF-α), nuclear factor kappa B (NF-κB), interleukin-1β (IL-1β), interleukin-6 (IL-6), c-Jun NH2-terminal kinase (JNK), mitogen-activated protein kinase 14 (MAPK14), MAPK15, receptor for advanced glycation endproducts (RAGE) and nod-like receptor family pyrin domain-containing 3 (NLRP3) in the liver tissue. Also produced endoplasmic reticulum stress by raising the mRNA transcript levels of activating transcription factor 6 (ATF-6), protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and glucose-regulated protein 78 (GRP-78). These factors together led to inflammation, apoptosis, and endoplasmic reticulum stress. On the other hand, liver tissue treated with ZN at doses of 25 and 50 mg/kg showed significant improvement in oxidative stress, inflammation, apoptosis and endoplasmic reticulum stress., Conclusions: Overall, the study's data suggest that administering ZN may be able to lessen the liver damage caused by SA toxicity., 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. The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Myeloid cells are the first line of defence against pathogens. Mitochondrial apoptosis signalling is a crucial regulator of myeloid cell lifespan and modulates the function of myeloid cells. The anti-apoptotic protein BCL-2-family protein BCL2A1/A1/BFL-1 is strongly upregulated in inflammation in macrophages. We analysed the contribution of A1 to apoptosis regulation in a conditional system of in vitro differentiation of murine macrophages from immortalised progenitors. We disabled the expression of A1 by targeting all murine A1 isoforms in the genome. Specific inhibitors were used to inactivate other anti-apoptotic proteins. Macrophage progenitor survival mainly depended on the anti-apoptotic proteins MCL-1, BCL-X L and A1 but not BCL-2. Deletion of A1 on its own had little effect on progenitor cell survival but was sensitised to cell death induction when BCL-X L or MCL-1 was neutralised. In progenitors, A1 was required for survival in the presence of the inflammatory stimulus LPS. Differentiated macrophages were resistant to inhibition of single anti-apoptotic proteins, but A1 was required to protect macrophages against inhibition of either BCL-X L or MCL-1; BCL-2 only had a minor role in these cells. Cell death by neutralisation of anti-apoptotic proteins completely depended on BAX with a small contribution of BAK only in progenitors in the presence of LPS. A1 and NOXA appeared to stabilise each other at the posttranscriptional level suggesting direct binding. Co-immunoprecipitation experiments showed the binding of A1 to NOXA and BIM. Interaction between A1 and Noxa may indirectly prevent neutralisation and destabilization of MCL-1. Our findings suggest a unique role for A1 as a modulator of survival in the macrophage lineage in concert with MCL-1 and BCL-X L , especially in a pro-inflammatory environment., (© 2024. The Author(s).)

This study aimed to determine the effect of ozone on the expression of Bax and Bcl-2 genes in dental pulp cells. Additionally, the programmed cell death protein 1, programmed death-ligand 1, and CD200 antigens were determined in lymphocytes to assess their surface expression. Dental pulp cells were cultured from extracted healthy third molars and characterized as dental pulp stromal cells. Gene expression of Bcl-2 and Bax was analyzed at 0 s, 6 s, and 12 s of ozone exposure using real-time PCR. Lymphocytes from dental pulp were subjected to ozone exposure for 12 s and PD-1, PD-L1, and CD200/CD200R expression was analyzed by flow cytometry. Upon exposure to ozone for 6 s, the Bcl-2 expression decreased significantly to -0.09, and at 12 s, it increased significantly to 0.3. Bax gene expression level increased significantly to 0.188 after 6 s exposure, and at 12 s, to 0.16. Lymphocytes exposed to ozone for 12 s showed minimal changes in PD-1, PD-L1, and CD200/CD200R expression levels, indicating that oxidative stress does not impact the signaling pathways regulating these molecules. The significant upregulation of Bcl-2 at 12 s highlights the cells' effort to protect themselves from prolonged oxidative stress, possibly tipping the balance toward cell survival and tissue repair. However, the absence of changes in PD-1 and PD-L1 expression on lymphocytes under oxidative stress suggests that these molecules are not sensitive to oxidative stress in this context., (© 2024 Maja Ptasiewicz et al., published by Sciendo.)

From previous studies, it is evident that metal-organic gold(I) complexes have antiproliferative activities. The aim of this study is not only to find new anticancer agents but also to overcome existing cytostatic resistance in cancer cells. The synthesis and medicinal evaluation of two cationic 1,3-disubstituted gold(I) bis-tetrazolylidene complexes 1 and 2 are reported. To determine apoptosis-inducing properties of the complexes, DNA fragmentation was measured using propidium iodide staining followed by flow cytometry. Gold(I) complex 1 targets explicitly malignant cells, effectively inhibiting their growth and selectively inducing apoptosis without signs of necrosis. Even in cells resistant to common treatments such as doxorubicin, it overcomes multidrug resistance and sensitizes existing drug-resistant cells to common cytostatic drugs. It is assumed that gold(I) complex 1 involves the mitochondrial pathway in apoptosis and targets members of the BCL-2 family, enhancing its potential as a therapeutic agent in cancer treatment.

The recombinant adeno-associated virus (rAAV) vector is among the most promising viral vectors in gene therapy. However, the limited manufacturing capacity in human embryonic kidney (HEK) cells is a barrier to rAAV commercialization. We investigated the impact of endoplasmic reticulum (ER) protein processing and apoptotic genes on transient rAAV production in HEK293 cells. We selected four candidate genes based on prior transcriptomic studies: XBP1, GADD34 / PPP1R15A, HSPA6, and BCL2. These genes were stably integrated into HEK293 host cells. Traditional triple-plasmid transient transfection was used to assess the vector production capability and the quality of both the overexpressed stable pools and the parental cells. We show that the overexpression of XBP1, HSPA6, and GADD34 increases rAAV productivity by up to 100% and increases specific rAAV productivity by up to 78% in HEK293T cells. Additionally, more prominent improvement associated with ER protein processing gene overexpression was observed when parental cell productivity was high, but no substantial variation was detected under low-producing conditions. We also confirmed genome titer improvement across different serotypes (AAV2 and AAV8) and different cell lines (HEK293T and HEK293); however, the extent of improvement may vary. This study unveiled the importance of ER protein processing pathways in viral particle synthesis, capsid assembly, and vector production. KEY POINTS: • Upregulation of endoplasmic reticulum (ER) protein processing (XBP1, HSPA6, and GADD34) leads to a maximum 100% increase in rAAV productivity and a maximum 78% boost in specific rAAV productivity in HEK293T cells • The enhancement in productivity can be validated across different HEK293 cell lines and can be used for the production of various AAV serotypes, although the extent of the enhancement might vary slightly • The more pronounced improvements linked to overexpressing ER protein processing genes were observed when parental cell productivity was high, with minimal variation noted under low-producing conditions., (© 2024. The Author(s).)

Purpose: We found a novel lncRNA named lncAC138150.2 related to the overall survival and staging of patients with colorectal cancer (CRC) by bioinformatic analysis using data from the Cancer Genome Atlas (TCGA), and the study aimed to elucidate the function of lncAC138150.2 and underlying mechanisms., Methods: Target molecules were knocked down by transfection with antisense oligonucleotides (ASOs), siRNAs, or lentiviruses and overexpressed by transfection with plasmids. The function of lncAC138150.2 was determined using histological, cytological, and molecular biology methods. The underlying mechanism of lncAC138150.2 function was investigated using RNA-seq, bioinformatics analysis, and molecular biology methods., Results: The expression of lncAC138150.2 was increased in colorectal tissues compared with paired normal tissues. The lncAC138150.2 knockdown increased apoptosis but did not change the cell proliferation, cell cycle distribution, or cell migration ability of CRC cells, while lncAC138150.2 overexpression decreased CRC apoptosis. lncAC138150.2 was mainly located in the cell nucleus, and each lncAC138150.2 transcript knockdown increased CRC apoptosis. BCL-2 pathway was significantly altered in apoptosis induced by lncAC138150.2 knockdown, which was alleviated by BAX knockdown. The expression of LYN was significantly decreased with lncAC138150.2 knockdown, LYN knockdown increased CRC apoptosis, and its overexpression completely alleviated CRC apoptosis induced by lncAC138150.2 knockdown., Conclusion: lncAC138150.2 significantly inhibited CRC apoptosis and affected the prognosis of patients with CRC, through the LYN/BCL-2 pathway., 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 Inc. All rights reserved.)

Objectives: Recent studies have shown that oxidative stress, inflammation, and apoptosis play substantial roles in the pathogenesis of tissue damage and distant organ damage during ovarian transplantation. Because of their potential antioxidant and anti-inflammatory properties, investigating natural compounds like curcumin and gallic acid is crucial. This study investigated the effects of curcumin, gallic acid, and their combination on liver and kidney tissues in a rat model of ovarian transplantation., Materials and Methods: For this study, we used 42 adult female Sprague-Dawley rats aged 11 to 12 weeks, which we randomly divided into 6 groups. After the transplant procedures, we performed histopathological analysis, biochemical examinations, and statistical analysis., Results: Significant damage was shown in the liver and kidney tissues of the ovarian transplant only and transplant plus corn oil groups compared with the control group, as evidenced by histopathological evaluation, increased BAX/BCL-2 intensity indicating apoptotic activity, and elevated interleukin 6 levels. However, treatment with curcumin, gallic acid, or their combination attenuated these adverse effects, suggesting potential protective effects against transplant-induced injury., Conclusions: Our findings highlight the therapeutic potential of these compounds in mitigating tissue damage and inflammation associated with ovarian transplant.

Glioblastoma multiforme is the most aggressive and invasive primary brain tumor in adults and its prognosis and survival rate remain poor. Despite substantial improvements in therapy, the 5-year survival rate of glioblastoma patients remains low. Sesquiterpenes have previously been found to be effective in inhibiting the proliferation and growth of breast, gastric and lung cancer cells. Owing to their efficacy, sesquiterpenes have been used in various clinical trials. In the present study, we investigated the anticancer efficacy of a well-known sesquiterpene, Zingiberene, isolated from Zingiber officinale in C6 glioblastoma cells. Zingiberene suppresses the growth and proliferation of C6 cells. Upon treatment of C6 cells with zingiberene, nuclear fragmentation and ROS were qualitatively enhanced compared to untreated control cells. The levels of caspase-3 were also significantly reduced (p<0.01), with a concomitant decline in the mRNA expression of Bax and Bcl-2. On the basis of molecular docking studies, Zingiberene demonstrated good binding energy score of -6.8 and -5.5 Kcal/mol towards Bax and Bcl-2 proteins, respectively. Based on these observations, it was inferred that zingiberene has potential as a plausible therapeutic agent against glioblastoma cells. Detailed mechanistic studies are needed to substantiate and establish the anticancer effects of zingiberene against glioblastoma cells.

Objective: Neoadjuvant chemotherapy (NACT) is widely used for treating locally advanced Breast cancer (LABC). However, development of multidrug resistance (MDR) is the main underlying factor for chemoresistance. Technetium-99m methoxyisobutylisonitrile (99mTc-MIBI) is a substrate for MDR. This study aimed to analyze the relationship between expression of MDR-related proteins (P-gp and Bcl-2) and 99mTc-MIBI uptake and retention in BC tumor cells, pathologic response to NACT, disease free survival (DFS) and overall survival (OS)., Methods: prospective analysis recruited 31 patients with LABC who received NACT between January 2019 and March 2020. 99mTc-MIBI planar and SPECT/CT imaging was conducted before and after NACT. Qualitative and quantitative analyses were performed, pre and post-NACT early and delayed lesion to non-lesion (LNL) ratios, and retention index (RI) of 99mTc-MIBI were calculated. Expression of P-gp and Bcl-2 in tumor cells was determined by immunohistochemistry., Results: Quantitively, inter-reader ICC for SPECT/CT based quantification was consistently higher than that of planar images. Post-NACT LNL ratios were significantly higher in patients with pathologic persistent disease (PPD). A change in RI between pre- and post-NACT scans demonstrated a significant association with DFS with a hazard ratio of 0.7 (95%CI: 06-1.0). Qualitatively, SPECT/CT was significantly more accurate compared to planar imaging in identifying residual viable tumor (81% compared to 57%).  Her2neu positivity and high post-operative Bcl-2 and P-gp were associated with worse DFS. A significant association was found between increased expression of post-NACT Bcl-2 and PPD, advanced tumor stage and poor OS., Conclusion: 99mTc-MIBI SPECT/CT based qualitative evaluation of BC response to NACT is more accurate than planar imaging. Post-NACT MIBI retention is positively correlated with P-gp and Bcl-2 expression. 99mTc-MIBI SPECT/CT may predict MDR development. High post-NACT Bcl-2 expression is significantly associated with advanced tumor stage and OS. High post-NACT P-gp expression has a worse impact on pathologic response and DFS.

Acute kidney injury (AKI) is a multifactorial syndrome with complex pathophysiology and prognosis. Ischaemia‒reperfusion injury (IRI) is a major cause of induced AKI. The aim of this study was to investigate the effect of upregulated CXCR7 expression on renal tubular epithelial cell apoptosis induced by hypoxia/reoxygenation (H/R). HK-2 cells were divided into three groups: control group (pcDNA3.1), hypoxia/reoxygenation + pcDNA3.1 group (H/R+pcDNA3.1) and CXCR7 overexpression + hypoxia/reoxygenation group (H/R+ Flag-CXCR7). Protein levels of renal tubular epithelial cell injury-, apoptosis- and autophagy-related markers were assessed by qRT‒PCR, Western blotting, flow cytometry (FCM), immunofluorescence and transmission electron microscopy (TEM). In addition, HK-2 cells were treated with the autophagy inhibitor 3-MA and divided into 3 groups: control group, 3-MA + pcDNA3.1 group, and 3-MA + Flag-CXCR7 group. Changes in autophagy and apoptosis in renal tubule epithelial cells were assessed by Western blotting and FCM. Compared with those in the control group, the protein and mRNA expression levels of CXCR7 in HK-2 cells were significantly lower under H/R conditions. Under H/R conditions, CXCR7 overexpression in HK-2 cells significantly downregulated the expression of NGAL. Moreover, CXCR7 overexpression significantly decreased H/R-induced cleaved PARP-1 and cleaved Caspase 3 levels, increased the level of the antiapoptotic protein BCL-2 and the autophagy-related molecules ATG5 and LC3B II, and significantly inhibited the expression of P62. Autophagy flow and TEM also showed that CXCR7 significantly promoted autophagy. CXCR7 significantly alleviated the 3-MA-induced inhibition of autophagy and increase in apoptosis. Upregulated CXCR7 expression can inhibit renal tubular epithelial cell apoptosis and damage by regulating autophagy. In conclusion, CXCR7 is a promising target for the prevention and/or treatment of AKI., (© 2024. The Author(s).)

Doxorubicin (Dox), an anthracycline antibiotic, is widely used in cancer treatment. Although its antitumor efficacy appears significant, its clinical use is greatly restricted by its induction of cardiotoxicity. Cardiac senescence drives the Dox-induced cardiotoxicity, but whether diminishing these senescent cardiomyocytes could alleviate the cardiotoxicity remains unclear. Here, we assessed whether senescent cardiomyocytes have a senescence-associated secretory phenotype (SASP) that affects healthy non-senescent cardiomyocytes, rendering them senescent via the delivery of exosomes. Additionally, we explored whether targeting SASP senescent cardiomyocytes using a Bcl-2 inhibitor could alleviate cardiotoxicity. Cardiac damage was induced in a mouse model of continuous Dox treatment in vivo, and cardiomyocytes in vitro. Immunofluorescence of the senescence markers of Cdkn2a, Cdkn1a and γ-H 2 A.X was used to assess the SASP in the Dox-treated heart. To explore the molecular mechanisms involved, the Bcl-2 inhibitor ABT-199 was employed to eliminate SASP senescent cardiomyocytes. We show that the cardiomyocytes acquire a SASP during Dox treatment. The senescent cardiomyocytes upregulated Bcl-2, although treatment of mice with ABT-199 selectively eliminated SASP senescent cardiomyocytes involved in Dox-induced cardiotoxicity, thus leading to partial alleviation of Dox-induced cardiotoxicity. Moreover, we concluded that SASP factors secreted by senescent cardiomyocytes induced by Dox renders otherwise healthy cardiomyocytes senescent through exosome delivery. Our findings suggest that SASP senescent cardiomyocytes are a significant component of the pathogenesis of Dox-dependent cardiotoxicity and indicate that targeting the clearance of SASP senescent cardiomyocytes could be a new therapeutic approach for Dox-induced cardiac injury., (© 2024 Federation of European Biochemical Societies.)

Purpose: We investigated the inhibitory effect of edaravone (EDR) lotion on chemotherapy-induced alopecia (CIA) to improve the quality of life for patients with cancer., Methods: Wistar rats were intraperitoneally injected with cyclophosphamide (CPA, 75 mg/kg) to induce CIA and divided into six groups: (1) Control; (2) EDR 0%; (3) EDR 0.3%; (4) EDR 3%. The TUNEL-positive area was examined histologically, and mRNA expression levels of the apoptosis-related factors, such as B-cell/CLL lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax), were determined., Results: In the three CPA-treated groups, a decrease in the coverage score (percentage of hairs covered) was observed from days 16 to 18. In addition, coverage scores on day 21, the last day of observation, showed a tendency for the suppression of hair loss to increase, though hair loss was observed in all groups. The coverage scores of the EDR 0.3% and 3% groups after day 17 were significantly higher than those of the EDR 0% group. The TUNEL-positive area of skin tissue on day 16 was extensive in the EDR 0% group and decreased in the EDR 0.3% and 3% groups. The mRNA expression ratio of Bcl-2/Bax on day 21 was maintained at the same level as that of the control group only in the EDR 3% group., Conclusion: This study confirmed the use of EDR lotion to inhibit hair loss, indicating that the clinical application of EDR lotion may improve the quality of life for patients with cancer and their willingness to undergo treatment., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mitochondria are cellular factories for energy production, calcium homeostasis and iron metabolism, but they also have an unequivocal and central role in intrinsic apoptosis through the release of cytochrome c. While the subsequent activation of proteolytic caspases ensures that cell death proceeds in the absence of collateral inflammation, other phlogistic cell death pathways have been implicated in using, or engaging, mitochondria. Here we discuss the emerging complexities of intrinsic apoptosis controlled by the BCL-2 family of proteins. We highlight the emerging theory that non-lethal mitochondrial apoptotic signalling has diverse biological roles that impact cancer, innate immunity and ageing. Finally, we delineate the role of mitochondria in other forms of cell death, such as pyroptosis, ferroptosis and necroptosis, and discuss mitochondria as central hubs for the intersection and coordination of cell death signalling pathways, underscoring their potential for therapeutic manipulation., (© 2024. Springer Nature Limited.)

Multiple myeloma (MM) is an incurable plasma cell malignancy that, despite an unprecedented increase in overall survival, lacks truly risk-adapted or targeted treatments. A proportion of patients with MM depend on BCL-2 for survival, and, recently, the BCL-2 antagonist venetoclax has shown clinical efficacy and safety in t(11;14) and BCL-2 overexpressing MM. However, only a small proportion of MM patients rely on BCL-2 (approx. 20%), and there is a need to broaden the patient population outside of t(11;14) that can be treated with venetoclax. Therefore, we took an unbiased screening approach and screened epigenetic modifiers to enhance venetoclax sensitivity in 2 non-BCL-2 dependent MM cell lines. The demethylase inhibitor 5-azacytidine was one of the lead hits from the screen, and the enhanced cell killing of the combination was confirmed in additional MM cell lines. Using dynamic BH3 profiling and immunoprecipitations, we identified the potential mechanism of synergy is due to increased NOXA expression, through the integrated stress response. Knockdown of PMAIP1 or PKR partially rescues cell death of the venetoclax and 5-azacytidine combination treatment. The addition of a steroid to the combination treatment did not enhance the cell death, and, interestingly, we found enhanced death of the immune cells with steroid addition, suggesting that a steroid-sparing regimen may be more beneficial in MM. Lastly, we show for the first time in primary MM patient samples that 5-azacytidine enhances the response to venetoclax ex vivo across diverse anti-apoptotic dependencies (BCL-2 or MCL-1) and diverse cytogenetic backgrounds. Overall, our data identify 5-azacytidine and venetoclax as an effective treatment combination, which could be a tolerable steroid-sparing regimen, particularly for elderly MM patients.

The Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins can mediate Bcl-2 interactions. We developed a highly-specific split luciferase assay enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a peculiar pro-apoptotic Bcl-2 protein. BOK-TMD and BCL-2-TMD interact at the endoplasmic reticulum. Molecular dynamics simulations confirm dynamic BOK-TMD and BCL-2-TMD dimers and stable heterotetramers. Mutation of BCL-2-TMD at predicted key residues abolishes interaction with BOK-TMD. Also, inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target., (© 2024. The Author(s).)