Cell Death Discovery

Ferroptosis has attracted significant attention for its potential to treat drug resistant tumors, along with its role in neurodegeneration and ischemia. Several ferroptosis targets were identified in the past decade, yet it is also highly affected by cellular metabolism and epithelial or mesenchymal phenotypes which limits the utility of these targets across diverse cell and tissue types. To address this, we utilized chemo-resistant cancer cell lines with different metastatic potential and tissue origins and discovered a common role of cytoplasmic ion changes during ferroptosis. We observed significant changes in intracellular potassium, sodium and chloride levels upon induction of ferroptosis. Therefore, we screened several ion channel inhibitors and activators (i.e. modulators) and identified Kv7.2 and CFTR modulators influence ferroptosis execution despite the fact that these cell lines do not express these proteins. We demonstrated that clinically approved modulators of CFTR and Kv7.2 can inhibit ferroptosis which indicates that these inhibitors could be repurposed as treatments in ferroptosis-associated diseases such as neurodegeneration and kidney diseases independent of ion channel expression. These modulators also influenced potassium and chloride levels in different contexts indicating off-target effects may also lead to changes in ion levels and may serve as a ferroptosis target. Further, we demonstrated that sustained imbalance of sodium, potassium and chloride ions with ferroptosis inducer salinomycin and digoxin, a Na+/K+ ATPase inhibitor, enhanced ferroptosis. The combination of salinomycin and digoxin successfully enhanced ferroptotic cell death across both cell lines with different metabolic signatures in vitro, offering a novel ferroptosis target. This combination significantly reduced tumor growth in vivo compared with the single agent alone, offering a new therapeutic modality against drug resistant tumors.

Palmitate-associated lipotoxic stress activates the endoplasmic-reticulum stress sensor protein IRE1, but how IRE1 signaling influences genotoxic-stress responses in triple-negative breast cancer remains unclear. Here, we investigated IRE1 and XBP1 signaling in MDA-MB-231 cells exposed to palmitate and etoposide. Under combined lipotoxic and genotoxic stress, XBP1 depletion increased DNA-damage accumulation in IRE1 wild-type cells but not in IRE1-deficient cells, whereas IRE1 re-expression reduced DNA damage. Pharmacologic IRE1 RNase inhibition also increased etoposide-associated DNA damage, supporting a protective role for IRE1 RNase-dependent signaling. IRE1 status and XBP1 manipulation differentially affected H2A.X and {gamma}H2A.X abundance, indicating context-dependent regulation of chromatin-associated DNA-damage signaling. At the RNA level, H2A.X was enriched in IRE1 immunoprecipitates, and the H2A.X-linked DNA-damage-response transcripts HUWE1 and BRCA2 also associated with IRE1 under combined stress. These findings link palmitate-activated IRE1-XBP1 signaling to genome-protective responses during genotoxic stress and identify a network of IRE1-associated DNA-damage-response transcripts in triple-negative breast cancer cells. Direct IRE1-mediated cleavage of these transcripts remains to be established.

Breast cancer progression is driven by dynamic changes in epithelial plasticity, membrane organization, and intracellular signaling, yet the effects of sustained low-dose chemotherapy on these processes remain poorly understood. Here, we investigated the impact of prolonged low-dose doxorubicin on membrane remodeling, epithelial phenotype, membrane-associated Ras lipid-anchor localization, and autophagy in mesenchymal-like MDA-MB-231 breast cancer cells. Low-dose doxorubicin significantly increased Caveolin-1 expression and enhanced E-cadherin protein levels, accompanied by a transition toward a more compact epithelial-like morphology with increased cell-cell contacts. Live-cell imaging demonstrated a significant reduction in the membrane-to-cytoplasm fluorescence ratio of the lipid-anchored GFP-tH probe, indicating redistribution from the plasma membrane to the cytoplasm following treatment. Analysis of autophagy-related proteins revealed decreased LC3-I together with increased LC3-II, ATG5, and p62 expression, consistent with autophagosome accumulation and impaired autophagic flux. Collectively, our findings demonstrate that low-dose doxorubicin promotes extensive remodeling of plasma membrane organization, epithelial plasticity, membrane-associated lipid-anchor localization, and autophagy. This integrated response reveals previously unrecognized links between membrane architecture, Ras membrane association, and autophagy during phenotypic reprogramming of breast cancer cells, providing mechanistic insight into cellular adaptations elicited by sub-cytotoxic doxorubicin exposure.

Pannexin 1 (PANX1) is a channel-forming glycoprotein that has been implicated in regulating melanoma cell properties such as growth, migration, and invasion. Here we report that the CRISPR-Cas9 mediated ablation of PANX1 or channel inhibition with probenecid shifted A375-P cell cycle distribution towards G2/M, while channel inhibition with spironolactone stalled cell cycle progression at the G1/S checkpoint regardless of PANX1 expression. Bulk RNA sequencing revealed cell cycle related gene expression was downregulated with PANX1 targeting strategies and this was exacerbated when PANX1 deficient cells were treated with spironolactone. When grown as spheroids, we found the deletion and inhibition of PANX1 disrupted spheroid compaction, with spheroids displaying poorly defined borders and reduced circularity. The deletion of PANX1 increased spheroid fragmentation, and this phenotype was intensified when spheroids devoid of PANX1 were treated with spironolactone, highlighting a potential synthetic lethal effect. Using liquid chromatography mass spectrometry, we sought to determine whether PANX1 had binding partners that may act to stabilize spheroid integrity and found that PANX1 co-immunoprecipitated various cell adhesion molecules such as {beta}-actin and F-actin-capping protein subunit -1, suggesting PANX1 and its interacting partners may stabilize cell-cell contacts. When control and PANX1 ablated cells were grown in chick chorioallantoic membrane xenografts to assess their tumour growth characteristics, we discovered that the deletion of PANX1 reduced tumour surface area and viability. Our findings indicate that PANX1 regulates cell cycle progression and the structural architecture of A375-P melanoma tumours, highlighting the therapeutic utility of targeting PANX1 in melanoma.

In our past study on human cilia from primary ciliary dyskinesia (PCD) patients using cryo-electron tomography and mass spectrometry, defects in the DNAH5 gene were proven to cause the loss of the entire outer dynein arm along with other proteins. This phenomenon was not observed in the unicellular green alga Chlamydomonas. In this study, we examined the loss of ciliary components caused by defects in various genes, based on proteomic and structural analyses of motile cilia from multiple PCD patients. The absence of single outer arm dynein genes causes a loss or significant decrease in other inner and outer arm dyneins. Interestingly, some patients with outer dynein defects show decreased intraflagellar transport (IFT) proteins, suggesting an influence of cargo components or assembly on transport. These phenomena were not observed in Chlamydomonas, suggesting a more complex mechanism of ciliogenesis in humans despite the high similarity in the final 3D architecture of the 9+2 axoneme. Defects in certain central pair proteins unexpectedly reduced components on the doublets as well. Our results demonstrate protein-protein interactions during stages of human motile ciliogenesis that are more complex than those in unicellular organisms.

As the main microtubule network organisers of animal cells, centrosomes orchestrate diverse cellular processes. Centrosomes have been linked to cancer when numerically or structurally aberrant, yet their precise roles at different cancer stages remains to be defined. Here, we study centrosome function in the epithelial-to-mesenchymal transition (EMT), a key driver of cancer initiation, progression and metastasis. We first depleted centrosomes in a breast cancer mesenchymal-like model, resulting in insignificant cellular or nuclear rearrangements. We then investigated non-carcinogenic breast cells undergoing the EMT via TGF{beta} signalling, and removed the centrosomes before triggering this transition. Stimulated epithelial cells acquired a mesenchymal phenotype and rearranged their cytoskeleton. By contrast, stimulated cells lacking centrosomes retained epithelial morphology, showed little cytoskeletal remodelling, yet still altered nuclear shape and upregulated key EMT markers. Transcriptome analysis revealed reduced expression of important genes involved in extracellular matrix organisation and a downregulation of cell junction components when lacking centrosomes; further analysis revealed paxillin showing reduced colocalisation with actin focal adhesions. Centrosomes thus prove essential for cytoskeletal remodelling in the EMT of breast cancer cells.

Protein misfolding in the endoplasmic reticulum (ER) of podocytes/glomerular epithelial cells (GECs) induces ER stress and contributes to the pathogenesis of glomerular disease. Activation of IRE1 and consequently the unfolded protein response (UPR) and autophagy sustains proteostasis. The present study examines the role of Surf4 and collagen IV-5 (COL4A5) trafficking in podocyte proteostasis. Glomerular Surf4 protein expression increased in IRE1-replete mice with experimental focal segmental glomerulosclerosis (FSGS) compared to podocyte-IRE1 knockout mice with FSGS and control mice. Glomerular Surf4 gene expression was greater in human FSGS and membranous nephropathy compared to healthy controls. In cultured GECs, basal Surf4 expression was greater in IRE1-replete (control) compared to IRE1 knockout GECs, although ER stress (tunicamycin treatment) did not increase Surf4 despite activating the UPR. Tunicamycin enhanced autophagy (LC3-II) in an IRE1-dependent manner, and the amount of Surf4 localized in autophagosomes. In GECs, tunicamycin increased the amount and colocalization of endogenous COL4A5 or ectopically-expressed COL4A5-wild type (WT) and Surf4 in autophagosomes. Compared to COL4A5-WT, the human COL4A5-C1567R mutant (which is misfolded) showed enhanced basal colocalization with Surf4 in autophagosomes; colocalization was similar to COL4A5-WT after tunicamycin treatment. Degradation of COL4A5-C1567R was more rapid compared to COL4A5-WT and was comparable to degradation of COL4A5-WT in the presence of tunicamycin. Thus, during ER stress, subsets of Surf4 and COL4A5 are redirected from the secretory pathway to autophagy. Surf4 may help deliver misfolded ER cargo, such as COL4A5 for autosomal degradation, thereby sustaining proteostasis in podocytes.

The association of cytoplasmic localization of VDR in TNBC tumors and disease aggressivity prompted investigation of its regulation by XPO1. In TNBC cell lines, the XPO1 inhibitors selinexor and eltanexor increased nuclear VDR localization. Their effects were particularly strong when combined with vitamin D treatment. Similarly, XPO1 knockdown increased nuclear VDR levels, both alone and in combination with vitamin D. These results identify XPO1 as an important regulator of VDR subcellular distribution. XPO1 inhibition also enhanced vitamin D-dependent VDR transcriptional activity. Indeed, selinexor, eltanexor, and XPO1 silencing increased vitamin D-dependent CYP24A1 reporter activity and the expression of genes such as CYP24A1 or CDH1. RNA sequencing showed that XPO1 modulation markedly reshaped the vitamin D transcriptional response. The affected genes were mainly involved in metabolism, cell-cycle regulation, protein localization, and cellular organization. Functional analyses demonstrated that cytoplasmic VDR promoted TNBC cell proliferation. Conversely, nuclear VDR was required for the antiproliferative effects of vitamin D. Accordingly, XPO1 inhibition or depletion potentiated vitamin D-mediated growth suppression. This finding suggested functional cooperation between XPO1-dependent export and VDR activity. Finally, high VDR expression was associated with poor disease-free or overall survival only in tumors with high XPO1 expression. Overall, XPO1 negatively regulates VDR nuclear localization and vitamin D responsiveness in TNBC. Targeting XPO1 may therefore enhance the therapeutic potential of vitamin D-based strategies in TNBC.

Hepatic stellate cells (HSCs) are the key cellular drivers of liver fibrosis. During liver injury and chronic inflammation HSCs adopt an activated phenotype and secrete fibrotic extracellular matrix (ECM) components such as collagen 1. Mechanical cues derived from the fibrotic ECM drive and support the activation of HSCs, via mechanisms that involve integrins and the mechano-sensitive transcriptional regulator YAP1. It is not yet well understood how external mechanical cues are translated into a molecular response that alters YAP1 nuclear shuttling. There is evidence that suggests the PDZ and LIM domain protein (PDLIM) 5 can regulate YAP1 shuttling in human epithelial cells. We therefore investigated whether PDLIM5 is expressed in HSCs and contributes to YAP1 associated HSC mechano-activation. PDLIM5 protein was detected in HSCs in fibrotic human and mouse liver. PDLIM5 transcript and protein were expressed by primary human and mouse HSCs and by the immortalised HSC LX-2 cell line. PDLIM5 localised with actin stress fibres suggesting a role in HSC adhesion. Co-immunoprecipitation and proximity ligation in LX-2 cells support an association between PDLIM5 and YAP1. We used pharmacological (paclitaxel) and genetic (siRNA and CRISPRi) approaches to inhibit PDLIM5 in HSCs. Inhibiting PDLIM5 reduced YAP1 nuclear localisation and fibrotic gene (COL1A1, ACTA2) expression in LX-2 cells. Overall, these data support a role for PDLIM5 in regulating YAP1 localisation and fibrogenic gene expression in HSCs.

Abstract Background Migrasomes, the newly discovered vesicular organelles, have long been speculated to originate from membrane dynamics during cell migration. Here we present integrated imaging and multi-omics evidence supporting a ciliary origin of migrasomes. The shared vesicular trafficking pathways between ciliogenesis and migrasome biogenesis (trans-Golgi/endosomal systems) prompted us to investigate their spatiotemporal coupling mechanism in disease models. Methods Using high-resolution SEM and fluorescence confocal microscopy (FCM), we visualized cilium-like structures with enlarged tips adjacent to migrasome-like vesicles across multiple cell types. TMT-based quantitative proteomics was used to identify proteins shared among migrasomes from different cell types and examine their associations with ciliary proteins. DMBA-induced breast cancer and femoral fracture models were used to investigate the cilia-migrasome association during tumor metastasis and fracture healing. Results Bioinformatics analysis showed a highly positive incidence correlation between cilia and migrasome genes in 31 types of 7858 human normal tissues and organs and various cell types. In 32 cancer tissues, cilia and migrasome genes were still strongly but abnormally correlated. Signaling enrichment by ARL13B and TSPAN4 showed that cilia-migrasome association is related to epithelial-mesenchymal transition (EMT). By analyzing the ARL13B and PIGK expression, we found the abnormal cilia-migrasome association in metastatic breast cancer and positive cilia-migrasome association in fracture healing bone. Conclusion Our findings support a ciliary origin of migrasomes, pointing to a previously unrecognized pathway of organelle biogenesis. Notably, the ciliary-migrasomal association correlates with two distinct pathological/physiological contexts: (1) aberrant epithelial-mesenchymal transition (EMT) in metastatic breast cancer, and (2) accelerated regeneration during fracture healing and tissue repair. This dual correlation not only establishes a mechanistic link between ciliary function and migrasome biogenesis but also highlights their divergent roles in disease progression versus tissue homeostasis. Investigating the spatiotemporal dynamics of this association may provide novel therapeutic targets for metastatic malignancies and regenerative medicine.

Triple-negative breast cancer (TNBC) is a highly aggressive malignancy and options for targeted therapy are limited. Although PARP inhibitors (PARPi) like olaparib (Ola) leverage synthetic lethality, their clinical utility is severely impeded by drug resistance and low frequency of BRCA mutations. In this study, 1,2,3,4,6-penta-O-galloyl-{beta}-D-glucose (PGG), a natural polyphenolic compound, can induce a state of homologous recombination deficiency (HRD) in TNBC cells by inhibiting the PALB2-BRCA2 interaction. PGG potentiated the inhibitory effects of Ola on the in vitro proliferation, clonogenic potential, migration, and invasion of TNBC cells. Mechanistically, the dual therapy triggered severe oxidative stress and mitochondrial collapse, which accelerated the classical apoptosis cascade and promoted the emission of immunogenic cell death (ICD)-associated DAMPs. The accumulated cytosolic double-stranded DNA activated the innate immune cGAS-STING pathway, thereby promoting robust intra-tumoral infiltration of CD8+ T cells. Furthermore, since cGAS-STING hyperactivation drives compensatory PD-L1 upregulation on TNBC cells, incorporating anti-PD-L1 antibody into the combination regimen further reversed the immunosuppressive barrier, achieving near-complete tumor eradication and maximum infiltration of cytotoxic T cells in vivo. Bioinformatics analyses further suggested a potential network of PGG targets associated with extracellular matrix organization, offering a bioinformatics-derived hypothesis for its theoretical involvement in modulating stromal tension and vascular homeostasis. Collectively, this study establishes PGG as a multi-functional therapeutic agent integrating intracellular synthetic lethality and innate immune sensing, while providing a preliminary bioinformatic rationale for prospective microenvironmental investigation. This study also shows PGG as a multi-functional therapeutic agent that integrates intracellular synthetic lethality, innate immune sensing, and microenvironmental remodeling to convert immunologically "cold" TNBC into "hot" tumors.

Colorectal cancer (CRC) exhibits substantial intratumoral heterogeneity, with differences in oncogenic driver mutations and apoptotic competence contributing to therapeutic resistance. Here, we investigated whether combined celecoxib and NDGA treatment (C+N) induces redox-mediated cell death via the p62/KEAP1 axis, offering a therapeutic approach that can overcome genetic diversity in CRC. We used two genetically distinct CRC cell lines, HCT116 and HT29, harbouring KRAS and BRAF mutations respectively, with differing p53 status, as models of tumor heterogeneity. C+N exhibited profound cytotoxicity in both cell lines, independent of COX-2/5-LOX expression and apoptotic competence. Additionally, C+N induced marked oxidative stress with mitochondrial ROS, lipid peroxidation, and labile ferrous iron accumulation, visualized as distinct JC-1 green mitochondrial puncta and FerroOrange red iron puncta in both cell lines. The antioxidant NAC reversed C+N-induced death, but inhibitors of apoptosis, autophagy, necroptosis, or ferroptosis did not, indicating a mechanism distinct from these pathways. Mechanistically, the combination induced p62 Ser349 phosphorylation, promoting KEAP1 degradation and NRF2 stabilization independently of ULK1-mediated autophagy; however, NRF2 stabilization was uncoupled from HO-1 induction. Our work reveals that C+N induces a ULK1/autophagy-independent, redox-mediated cell death mechanism that circumvents apoptotic competence, highlighting redox disruption as a genotype-independent therapeutic strategy to overcome CRC heterogeneity.

Spatiotemporal allocation and dynamic redistribution of centrosomal proteins are critical for coordinating the structural and molecular events that ensure successful cell division. While depletion of the multifunctional protein Optineurin (OPTN) impairs cell-cycle progression, our previous report of the presence of OPTN at key cell-division assemblies raised the question of how its trafficking is coordinated during cell division. In this study, using cell-cycle synchronisation coupled with flow cytometric analysis and confocal microscopy, we report that OPTN progressively dissociates from centrosomes during mitosis, redistributes to cytokinetic structures, accumulates at the spindle midzone, and then concentrates at the midbody during cytokinesis. Moreover, following this transition, OPTN progressively re-emerges at centrosomes during G1 of the subsequent cell cycle. Further, microtubule perturbation and regrowth assays provide evidence that OPTN recruitment and retention at the pericentriolar matrix (PCM) can occur independently of microtubules, whereas its exit is microtubule-dependent. Collectively, this study depicts, for the first time, a detailed movement trail of OPTN, highlighting its centrosome-linked redistribution and microtubule dependency. This context-dependent localisation and trafficking of OPTN across the centrosome-midbody axis establish a definitive spatiotemporal roadmap that can be strategically utilised to study its functional role in cell division.

Carbaryl, a widely used carbamate pesticide, has been epidemiologically associated with genotoxic and carcinogenic outcomes in exposed human populations, including elevated cancer risk at several tissue sites and, in some agricultural communities, chronic tubulointerstitial kidney disease of unknown etiology (CKDu). However, the cellular mechanisms underlying these associations, and whether carbaryl acts uniformly across tissues, remain poorly defined. Screening a panel of cell lines of distinct species and lineage, we found that carbaryl elicits pronounced, dose-dependent cytotoxicity and cell cycle arrest selectively in a renal tubular epithelial line (IMCD3) and, to a lesser extent, a cervical epithelial line (HeLa), while a fibroblast line (NIH3T3) was unaffected. In IMCD3 cells, carbaryl induces S- and G2/M-phase arrest through direct binding to DNA and consequent DNA damage, triggers Bcl-2-mediated late apoptosis independently of reactive oxygen species, and drives irreversible mitotic arrest. Molecular dynamics simulations, cellular thermal shift assays, and kinase measurements converge on CDK1 as a direct target of carbaryl: carbaryl destabilises the CDK1 active site and inhibits its catalytic activity, whereas CDK2 is engaged through an opposing, rigidifying mode with no functional consequence. This asymmetry provides a coherent mechanistic basis for the combined S-phase and G2/M arrest observed. Together, these findings establish carbaryl as a genotoxic, cell-cycle-disrupting agent with tissue-selective potency acting directly on CDK1, and provide a cell-based mechanistic framework for interpreting its reported associations with renal and other tissue pathologies in exposed human populations.

Intestinal epithelial barrier is a key feature of the healthy gut, and disruption of this barrier is a driver of mucosal inflammation. Integrity of the intestinal epithelial barrier is controlled by tight junctions (TJs) and adherens junctions (AJs) associated with the underlying actin cytoskeleton. Posttranslational modifications of actins affect the cytoskeletal assembly and dynamics, however whether such modifications regulate epithelial barriers remains unknown. SETD3 methyltransferase methylates actins at the His73 residue, thereby enhancing filamentous (F)-actin polymerization and turnover. This study examines the role of SETD3 in regulating structure and function of normal intestinal epithelial barrier, as well as its involvement in barrier disruption during mucosal inflammation. siRNA mediated knockdown of SETD3 in DLD-1 and HCA-7 human intestinal epithelial cells (IEC) resulted in several functional and morphological cell abnormalities. These include increased permeability of the IEC barrier, diminished assembly of the perijunctional F-actin belt, increased cell size, and delayed TJ reassembly during calcium switch. Consistently, SETD3 knockout (KO) mice displayed increased intestinal permeability and decreased labeling intensity of apical {beta}-actin as compared to wildtype controls. Furthermore, SETD3 KO mice showed increased cell stemness and proliferation in colonic epithelium without defects in AJ/TJ assembly and secretory cell differentiation. In addition, SETD3 KO mice developed more severe disease symptoms during dextran sodium sulfate (DSS)-induced colitis, which were accompanied by accelerated mucosal erosion and increased macrophage, T cell and neutrophil infiltration into colonic tissue. Overall, our data suggests that SETD3 acts as a novel positive regulator of the intestinal epithelial barrier and plays a protective role during intestinal mucosal damage and inflammation.

Abstract Background. Tuberous Sclerosis Complex (TSC) is caused by inactivating mutations in either the TSC1 or TSC2 genes, leading to activation of the mammalian target of rapamycin complex 1 (mTORC1) and unhindered cell growth and proliferation. The epithelium of TSC renal cysts in both mice and humans is composed of proliferating A-intercalated (A-IC) cells. The exact molecular mechanism of kidney cystogenesis in TSC remains speculative. Hypothesis.Superfluous cell proliferation driven by mTORC1 activation increases metabolic demand, causing oxidative stress and excess reactive oxygen species. If unchecked, this overwhelms antioxidant defenses and causes cell death. In TSC kidney cystogenesis, Nuclear Factor Erythroid 2-Related Factor 2 (NFE2L2), also known as NRF2, serves as the "master regulator" of antioxidant and anti-inflammatory responses, enabling cells to survive and proliferate by clearing the toxic environment and supplying nutrients and fuel. Results. RNA-seq and proteomics, along with western blot analysis, showed robust downregulation of Fumarate Hydrase 1 (FH1) and upregulation of NRF2, STAT3, and HIF1 in kidneys from TSC mice with moderate or heavy cyst burden. Confocal microscopy and immunohistochemical staining on kidney sections, and/or western blot studies on nuclear and cytoplasmic fractions, showed nuclear localization of NRF2, STAT3, and HIF1. In cyst-lining cells in TSC mouse models, FH1 downregulation was associated with inactivating succination of KEAP1 in immunoprecipitation experiments, promoting NRF2 nuclear localization in A-IC cells lining the cysts. NRF2 nuclear localization was associated with ectopic induction of the glutamine transporter SLC38A3 (SNAT3) on the basolateral membrane and activation of the NH3/NH4+ transporters RHCG and RHBG in A-IC cells lining the cysts. Twenty-four h urine NH3/NH4+ excretion rates increased significantly in Tsc1 KO vs. WT mice. The activation of NRF2, STAT3, and HIF1 can drive metabolic reprogramming and activate survival genes in proliferating cells. Together with SLC38A3 induction and upregulation of other glutamine and NH3/NH4+ transporters, these factors activate glutaminolysis and aerobic glycolysis, supplying nutrients to proliferating cystic epithelial cells and supporting cyst expansion in TSC. Consistent with this central role for glutaminolysis in kidney cystic epithelium and TSC cystogenesis, we find a significant reduction in kidney cyst burden in Tsc1 KO mice on a glutamine-free diet. Conclusions. NRF2 plays a critical role in antioxidant defense. Along with STAT3 and HIF1, NRF2 is a key player in metabolic reprogramming through glutaminolysis, which supplies nutrients to proliferating cystic epithelial cells and supports cyst expansion in TSC. These findings suggest that inhibiting or inactivating NRF2, alone or in combination with HIF1 or STAT3, may represent a potential treatment strategy for kidney lesions in TSC.

Although therapeutic advancements have dramatically improved outcomes for cancer patients in recent decades, advanced cancer remains a significant challenge. There is still an urgent need for new and more effective strategies that minimize toxicity and improve survival outcomes in cancer patients. Natural products from microorganisms are a rich and promising source of bioactive compounds with substantial therapeutic potential in cancer treatment. Recently, B. proteolyticus strain OSUB18, isolated from switchgrass, has been demonstrated to promote plant growth and regulate reactive oxygen species (ROS) production. Here, we investigated the anticancer potential of this plant-associated microbe and demonstrated that its crude extracts exhibited broad-spectrum cytostatic activity across diverse solid tumor cell lines. Importantly, OSUB18 extracts displayed limited cytotoxicity toward normal human mammary epithelial cells and mouse fibroblasts. This potentially selective cytostatic activity was retained within a water soluble, heat-labile, and protease-resistant fraction. The extracts primarily suppressed cell proliferation, accompanied by increased lipid peroxidation, with preliminary evidence suggesting the potential involvement of ferroptosis. These findings suggest that the bioactive component may be a selective cytostatic agent with multifaceted mechanisms of action.

Chemobrain, characterized by cognitive dysfunction, has emerged as a significant health concern following cancer treatment. Doxorubicin, an FDA-approved chemotherapeutic agent, is widely used to treat various cancers. However, its adverse effects raise concerns regarding toxicity in non-cancerous cell types. Microglia, the resident immune cells of the brain, play a central role in maintaining neural homeostasis. Despite this, the mechanisms by which microglia become dysfunctional following doxorubicin exposure remain poorly understood. Understanding the transcriptomic alterations induced by doxorubicin in microglia could provide critical insights into mechanisms underlying its neurotoxic effects. In this study, the transcriptomic response of human microglia to doxorubicin using mRNA sequencing combined with bioinformatic analysis was investigated. Exposure to doxorubicin significantly altered microglial morphology, promoting a shift toward a process-bearing phenotype in human HMC3 cells. This phenotypic change was accompanied by elevated levels of DNA damage and was associated with the induction of cellular senescence. At the gene regulation level, the Illumina-based transcriptomic analysis provided 25,834 gene expression profiling in which 5,669 were differentially expressed. The future analyses revealed that 3,431 (upregulated) and 2,238 (downregulated) were differentially expressed under the treatment of doxorubicin. Genes associated with the p53 signaling pathway and inflammatory responses were upregulated, whereas genes involved in translation, DNA replication, and cell-cycle progression were downregulated. This transcriptional dysregulation was accompanied by differential expression of key transcription factors. This study provides transcriptomic evidence elucidating the genotoxic response to doxorubicin in microglial cells, offering mechanistic insights into chemobrain and highlighting key molecular pathways as potential targets for therapeutic intervention.

Metacaspases are cysteine proteases and are found in every group of organisms except animals. They are structural orthologs of caspases, which orchestrate apoptosis in animals. Caspases, metacaspases, paracaspases and orthocaspases are class C14 proteases with a similar active site (the hemoglobinase fold) and a catalytic cysteine residue. However, metacaspases cleave after arginine and lysine instead of aspartate and some researchers point out that a name ending with caspase is unsuitable. Also, most metacaspases are activated by calcium, they do not target the same proteins as caspases and they cut multiply instead of once. The Candida albicans metacaspase Mca1p mediates cell death in response to various stresses but also has pro-life functions. Many plant and protist metacaspases lack cell death roles and are involved in development, differentiation and immunity. This article highlights an interesting phenomenon, whereby the promotion of stress sensitivity and reduction of virulence, by Mca1p in C. albicans cells from exponential culture are abolished and reversed, respectively in cells from stationary-phase culture. This hints at the proteases's dual nature and at the conditions that might initiate a switch between pro-life and pro-death functions.

Nucleolar alterations are characteristic features of cancer cells; however, how changes in the overall morphology relate to internal subcompartment organization and ribosomal RNA (rRNA) dynamics remains unclear. In this study, the nucleolar organization of non-transformed MCF-10A mammary epithelial cells and MDA-MB-231 breast cancer cells was quantitatively compared. MDA-MB-231 cells exhibited differences in nucleolar morphology, accompanied by altered spatial organization of the dense fibrillar component and granular component, and accelerated intranucleolar trafficking of nascent rRNA. Pharmacological perturbation with SU056 shifted multiple structural and dynamic parameters toward an MCF-10A-like profile. Conversely, transient physicochemical perturbation with 1,6-hexanediol induced several MDA-MB-231-like nucleolar features in MCF-10A cells, which returned to their untreated profile after washout. These findings support the view that cancer-associated nucleolar alterations are an integrated reorganization across morphological, spatial, and dynamic dimensions. The observed reversibility further indicates that cancer-associated nucleolar organization can be remodeled in response to perturbations. By linking the morphology, subcompartment organization, and nascent rRNA dynamics within a common framework, this study provides a basis for exploring the establishment, maintenance, and remodeling of distinct nucleolar profiles in cancer.