Search Results (74)

Background: The treatment landscape for multiple myeloma (MM) has significantly evolved in recent decades with novel therapies like proteasome inhibitors, immunomodulatory drugs and monoclonal antibodies. However, MM remains incurable, necessitating new pharmacological strategies. Mitotic kinases, such as Aurora proteins, have emerged as potential targets. Selective inhibitors of Aurora A and B,- alisertib (MLN8237) and barasertib (AZD1152), respectively, have shown anti-myeloma activity in preclinical studies, with alisertib demonstrating modest efficacy in early clinical trials. Methods and Results: This study investigated the mechanisms of action of alisertib and barasertib and their combination with antitumor agents in a panel of five MM cells lines. Both drugs induced cell cycle arrest phase and abnormal nuclear morphologies. Alisertib caused prolonged mitotic arrest, whereas barasertib induced transient arrest, both resulting in the activation of mitotic catastrophe. These findings revealed three potential outcomes: cell death, senescence, or polyploidy. High mitochondrial reactive oxygen species (mROS) were identified as possible drivers of cell death. Caspase inhibition reduced caspase-3 activation but did not prevent cell death. Interestingly, alisertib at low doses remained toxic to Bax/Bak^DKO cells, although mitochondrial potential disruption and cytochrome c release were observed. Sequential combinations of high-dose Aurora kinase inhibitors with BH3-mimetics, and in specific cases with panobinostat, showed a synergistic effect. Conversely, the simultaneous combination of alisertib and barasertib showed mostly antagonistic effects. Conclusions: Alisertib and barasertib emerge as potential in vitro candidates against MM, although further studies are needed to validate their efficacy and to find the best combinations with other molecules. Full article

One of the hallmarks of cancer cells is their failure to respond to the cellular mechanism of apoptosis. The B-cell lymphoma 2 (BCL-2) family of proteins regulate apoptosis. Their ability to do so can be measured using several methods that in turn anticipate the fate of the cancer cell in response to apoptosis-inducing treatment. These assays ultimately identify the readiness of the cancer cell to undergo apoptosis, which is referred to as the mitochondrial priming state. These metrics, however, have been challenging to implement in the clinic. Methods: Here, we describe a unique method that relies on a panel of novel conformation-specific antibodies (termed PRIMAB) that can directly measure the mitochondrial priming state. These reagents are highly specific for complexes of their corresponding pro-survival protein interactions with the pro-apoptotic protein BIM. These BIM-containing heterodimeric complexes have long been established as hallmarks of primed cancer cells. Results: Using clinically amenable assay formats, PRIMABs were shown to detect the presence of these anti-apoptotic–pro-apoptotic complexes and their disruption by BH3-mimetic drugs. Moreover, PRIMABs were able to detect a shift in priming status following BH3-mimetic treatment, a factor associated with resistance to these drugs. In a panel of AML patient samples, we report a wide range of priming levels for each PRIMAB complex, demonstrating the potential for heterogeneity in responses. We also show that PRIMABs could be predictive of outcomes for AML patients following cytarabine-based treatment. Conclusions: PRIMABs provide novel and useful tools for cancer research and for clinical implementation as reagents providing predictive tests for treatment response. Full article

Glioblastoma, classified as a grade IV astrocytoma, is an aggressive and malignant primary brain tumor with no known cure. Despite the implementation of standard medical and surgical treatment protocols, the disease often progresses with unsatisfactory outcomes. This study aimed to evaluate the cytotoxic, proapoptotic, and antimetastatic effects of anti-angiogenic monoclonal antibody bevacizumab combined with the sphingosine-1-phosphate receptor modulator fingolimod on rat glioma C6 cells. The cytotoxicity of bevacizumab and fingolimod was evaluated using the MTT assay. Proapoptotic activity was assessed through flow cytometric analyses, including Annexin V–FITC staining, caspase 3/7 activation, and mitochondrial membrane potential measurements. Morphological changes were examined using confocal microscopy. Antimetastatic effects were evaluated via anti-migration and colony formation assays. The combination of bevacizumab and fingolimod exhibited antiproliferative, cytotoxic, proapoptotic, and antimetastatic effects on C6 glioma cells at low IC₅₀ concentrations. Based on growth inhibitory, proapoptotic, and antimetastatic activities on C6 glioma cells, the combination of bevacizumab and fingolimod demonstrates significant growth-inhibitory, proapoptotic, and antimetastatic activities against C6 glioma cells, suggesting its potential as a promising pharmacotherapeutic approach for the treatment of glioblastoma. Full article

The pathogenesis of primary biliary cholangitis (PBC) is not fully understood. Despite recent progress, many aspects require further clarification. Thus, PBC is regarded as an autoimmune disease, but immunosuppressive treatment, which is effective in other autoimmune diseases, is not working in the case of PBC. Moreover, there are controversies over the pathogenetic role of anti-mitochondrial antibodies as mitochondria are present in all cells but only cholangiocytes are damaged. In this review, all the proposed models and factors that have been involved in the pathogenesis of PBC are presented. They include mechanisms such as dysregulated autophagy, senescence, apoptosis, impairment of the protective bicarbonate umbrella, immunological abnormalities, the dysbiosis of gut microbiota, and the role of bile acids. Genetics of PBC and epigenetic transcriptional modifications are also presented. Data supporting molecular mimicry and the viral etiology of PBC are analyzed. Finally, an integrated model is proposed based on interactions of the factors that may participate in PBC pathogenesis. Therefore, the purpose of this review is to provide a unifying presentation of the various aspects of PBC pathophysiology, which will allow for a better understanding of this multifaceted disease. New treatment targets may also be identified in such a holistic model. Full article

The disruption of microglial homeostasis and cytokine release are critical for neuroinflammation post-injury and strongly implicated in retinal neurodegenerative diseases like glaucoma. This study examines microglial responses to chemical hypoxia induced by cobalt chloride (CoCl₂) in BV-2 murine microglial cells, focusing on signaling pathways and proteomic alterations. We assessed the protective effects of monoclonal antibodies against TNFα and IL-1β. CoCl₂ exposure led to decreased cell viability, reduced mitochondrial membrane potential, increased lactate dehydrogenase release, elevated reactive oxygen species generation, and activation of inflammatory pathways, including nitric oxide synthase (iNOS), STAT1, and NF-κB/NLRP3. These responses were significantly mitigated by treatment with anti-TNFα and anti-IL-1β, suggesting their dual role in reducing microglial damage and inhibiting inflammatory reactivity. Additionally, these treatments reduced apoptosis by modulating ATF4 and the p38 MAPK/caspase-3 pathways. Label-free quantitative mass spectrometry-based proteomics and Gene Ontology revealed that CoCl₂ exposure led to the upregulation of proteins primarily involved in endoplasmic reticulum and catabolic processes, while downregulated proteins are associated with biosynthesis. Anti-TNFα and anti-IL-1β treatments partially restored the proteomic profile toward normalcy, with network analysis identifying heat shock protein family A member 8 (HSPA8) as a central mediator in recovery. These findings offer insights into the pathogenesis of hypoxic microglial impairment and suggest potential therapeutic targets. Full article

Mitochondria are important organelles for cell metabolism and tissue survival. Their cell-to-cell transfer is important for the fate of recipient cells. Recently, bone marrow mesenchymal stem cells (BM-MSCs) have been reported to provide mitochondria to cancer cells and rescue mitochondrial dysfunction in cancer cells. However, the details of the mechanism have not yet been fully elucidated. In this study, we investigated the humoral factors inducing mitochondrial transfer (MT) and the mechanisms. BM-MSCs produced MT in colorectal cancer (CRC) cells damaged by 5-fluorouracil (5-FU), but were suppressed by the anti-high mobility group box-1 (HMGB1) antibody. BM-MSCs treated with oxidized HMGB1 had increased expression of MT-associated genes, whereas reduced HMGB1 did not. Inhibition of nuclear factor–κB, a downstream factor of HMGB1 signaling, significantly decreased MT-associated gene expression. CRC cells showed increased stemness and decreased 5-FU sensitivity in correlation with MT levels. In a mouse subcutaneous tumor model of CRC, 5-FU sensitivity decreased and stemness increased by the MT from host mouse BM-MSCs. These results suggest that oxidized HMGB1 induces MTs from MSCs to CRC cells and promotes cancer cell stemness. Targeting of oxidized HMGB1 may attenuate stemness of CRCs. Full article

Osteoarthritis (OA) is the most common rheumatologic disease and a major cause of pain and disability in older adults. No efficient treatment is currently available. Mitochondrial dysfunction in chondrocytes drives molecular dysregulation in OA pathogenesis. Recently, mitochondrial transfer to chondrocytes had been described, enabling transplant of mitochondria as a new avenue to modify the OA process, although evidence on its feasibility and safety remains limited.The primary objective of this study was to demonstrate the feasibility and safety of intra-articular mitochondrial transplantation. Mitochondria were isolated from liver using the procedure described by Preble and coworkers combined with magnetic beads coupled to anti-TOM22 antibodies. The organelles obtained were analyzed to determine their purity and viability. The safety and viability of the administration of the isolated mitochondria into articular tissues as well as the integration and distribution of the transplanted mitochondria within joint tissues were analyzed using both in vitro and in vivo models. We established an efficient, reproducible, effective, and rapid protocol for isolating mitochondria from liver. We obtained mitochondria with high viability, yield, and purity. The isolated mitochondria were injected into joint tissue using both in vitro and in vivo models. Functional mitochondria were detected in the extracellular matrix of the cartilage, menisci and synovium. Our results establish a safe and viable protocol for mitochondrial isolation and intra-articular injection. The methodology and findings presented here pave the way for future studies in osteoarthritis models to validate mitochondrial transplantation as a potentially effective treatment for OA. Full article

Background and Objectives: A large amount of recent evidence suggests that cellular inability to consume oxygen could play a notable part in promoting sepsis as a consequence of mitochondrial dysfunction and oxidative stress. The latter could, in fact, represent a fundamental stage in the evolution of the “natural history” of sepsis. Following a study previously conducted by the same working group on heart samples, the present research project aims to evaluate, through an immunohistochemical study, the existence and/or extent of oxidative stress in the brains of subjects who died due to sepsis and define, after reviewing the literature, its contribution to the septic process to support the use of medications aimed at correcting redox anomalies in the management of septic patients. Materials and Methods: 10 cases of subjects who died in healthcare facilities with ante-mortem clinical-laboratory signs that allowed the diagnosis of septic shock were selected as case studies, and 1 case of a subject who died immediately following a road traffic accident was used as a negative control. Samples of the cerebral cortex were then taken, fixed in formalin, and subjected to sections on which an immunohistochemical study was performed using anti-NOX-2, NT, iNOS, and 8-OHdG antibodies. Results: The results emerging from the present study demonstrate that despite a variable expressivity for the NT, iNOS, and NOX2 markers, the brain samples demonstrated univocal and high positivity for the 8-OHdG marker. Conclusions: This would allow us to hypothesize how, regardless of the mechanism of production of ROS and NOS (iNOS or NOX2 mediated) and the pathophysiological mechanisms that are triggered during sepsis, oxidative damage to DNA represents the event to which this whole process leads and, in fact, in the literature, is directly correlated to sepsis-dependent mortality. Neurons, conversely, appear to be more sensitive to oxidative stress because of a low number of protective or scavenger molecules (catalase, glutathione peroxidase, GSH, or vitamin E). Therefore, despite reduced production, the manifestation of the damage remains high. This evidence, together with that of the previous study, can only support the introduction of substances with an antioxidant function in the guidelines for the treatment of sepsis. Full article

Gastric cancer remains a significant therapeutic challenge, highlighting the need for new strategies to improve treatment efficacy. This study investigates the potential of combined therapy with the novel Thiopyrano[2,3-d]Thiazole derivative LES-6400 and the anti-HER2 antibody trastuzumab in AGS gastric cancer cells. The antitumor effects of the combined therapy were evaluated using various techniques, including the MTT assay for cell viability, [³H]-thymidine incorporation for DNA synthesis, and flow cytometry to assess apoptosis (Annexin V-FITC/PI staining), mitochondrial membrane potential (MMP), and inflammatory cytokine levels. ELISA was employed to measure the levels of IL-6, p53, and cytochrome C. The combination of LES-6400 (1 µM) and trastuzumab (10 µg/mL) demonstrated superior antitumor activity compared to monotherapy with either agent in AGS gastric cancer cells. The combination therapy enhanced apoptosis, presumably by inducing oxidative stress in the cells and disrupting mitochondrial membrane potential. Additionally, a significant increase in p53 protein levels and modulation of interleukin levels, including a marked reduction in IL-6 levels, were observed, suggesting an impact on apoptotic and inflammatory responses. These findings indicate that the combined use of LES-6400 and trastuzumab is a promising therapeutic strategy for gastric cancer, warranting further investigation into the mechanisms of action and potential clinical applications of this combined approach. Full article

Background: The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to viral entry and can cause cardiac injuries. Toll-like receptor 4 (TLR4) and NOD-, LPR-, and pyrin-domain-containing 3 (NLRP3) inflammasome are critical immune system components implicated in cardiac fibrosis. The spike protein activates NLRP3 inflammasome through TLR4 or angiotensin-converting enzyme 2 (ACE2) receptors, damaging various organs. However, the role of spike protein in cardiac fibrosis in humans, as well as its interactions with NLRP3 inflammasomes and TLR4, remain poorly understood. Methods: We utilized scratch assays, Western blotting, and immunofluorescence to evaluate the migration, fibrosis signaling, mitochondrial calcium levels, reactive oxygen species (ROS) production, and cell morphology of cultured human cardiac fibroblasts (CFs) treated with spike (S1) protein for 24 h with or without an anti-ACE2 neutralizing antibody, a TLR4 blocker, or an NLRP3 inhibitor. Results: S1 protein enhanced CFs migration and the expressions of collagen 1, α-smooth muscle actin, transforming growth factor β1 (TGF-β1), phosphorylated SMAD2/3, interleukin 1β (IL-1β), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). S1 protein increased ROS production but did not affect mitochondrial calcium content and cell morphology. Treatment with an anti-ACE2 neutralizing antibody attenuated the effects of S1 protein on collagen 1 and TGF-β1 expressions. Moreover, NLRP3 (MCC950) and NF-kB inhibitors, but not the TLR4 inhibitor TAK-242, prevented the S1 protein-enhanced CFs migration and overexpression of collagen 1, TGF-β1, and IL-1β. Conclusion: S1 protein activates human CFs by priming NLRP3 inflammasomes through NF-κB signaling in an ACE2-dependent manner. Full article

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that comprises amyloid-beta protein (Aβ) as a main component of neuritic plaques. Its deposition is considered a trigger for AD pathogenesis, progression, and the clinical symptoms of cognitive impairment. Some distinct pathological features of AD include phosphorylation of tau protein, oxidative stress, and mitochondrial dysfunction. These pathological consequences tend to produce reactive oxygen species (ROS), resulting in the dysregulation of various signaling pathways of neuroinflammation and neurodegeneration. The relationship between the Aβ cascade and oxidative stress in AD pathogenesis is like a “chicken and egg” story, with the etiology of the disease regarding these two factors remaining a question of “which comes first.” However, in this review, we have tried our best to clarify the interconnection between these two mechanisms and to show the precise cause-and-effect relationship. Based on the above hallmarks of AD, several therapeutic strategies using natural antioxidants, monoclonal antibodies, and vaccines are employed as anti-Aβ therapy to decrease ROS, Aβ burden, chronic neuroinflammation, and synaptic failure. These natural antioxidants and immunotherapeutics have demonstrated significant neuroprotective effects and symptomatic relief in various in vitro and in vivo models, as well as in clinical trials for AD. However, none of them have received final approval to enter the drug market for mitigating AD. In this review, we extensively elaborate on the pitfalls, assurances, and important crosstalk between oxidative stress and Aβ concerning current anti-Aβ therapy. Additionally, we discuss future strategies for the development of more Aβ-targeted approaches and the optimization of AD treatment and mitigation. Full article

While cytostatic chemotherapy targeting DNA is known to induce genotoxicity, leading to cell cycle arrest and cytokine secretion, the impact of these drugs on fibroblast–epithelial cancer cell communication and metabolism remains understudied. Our research focused on human breast fibroblast RMF-621 exposed to nonlethal concentrations of cisplatin and doxorubicin, revealing reduced proliferation, diminished basal and maximal mitochondrial respirations, heightened mitochondrial ROS and lactate production, and elevated MCT4 protein levels. Interestingly, RMF-621 cells enhanced glucose uptake, promoting lactate export. Breast cancer cells MCF-7 exposed to conditioned media (CM) from drug-treated stromal RMF-621 cells increased MCT1 protein levels, lactate-driven mitochondrial respiration, and a significantly high mitochondrial spare capacity for lactate. These changes occurred alongside altered mitochondrial respiration, mitochondrial membrane potential, and superoxide levels. Furthermore, CM with doxorubicin and cisplatin increased migratory capacity in MCF-7 cells, which was inhibited by MCT1 (BAY-8002), glutamate dehydrogenase (EGCG), mitochondrial pyruvate carrier (UK5099), and complex I (rotenone) inhibitors. A similar behavior was observed in T47-D and ZR-75-1 breast cancer cells. This suggests that CM induces metabolic rewiring involving elevated lactate uptake to sustain mitochondrial bioenergetics during migration. Treatment with the mitochondrial-targeting antioxidant mitoTEMPO in RMF-621 and the addition of an anti-CCL2 antibody in the CM prevented the promigratory MCF-7 phenotype. Similar effects were observed in THP1 monocyte cells, where CM increased monocyte recruitment. We propose that nonlethal concentrations of DNA-damaging drugs induce changes in the cellular environment favoring a promalignant state dependent on mitochondrial bioenergetics. Full article

Mitochondrial dysfunction has been increasingly recognized as a trigger for systemic lupus erythematosus (SLE). Recent bioinformatics studies have suggested Fam210b as a significant candidate for the classification and therapeutic targeting of SLE. To experimentally prove the role of Fam210b in SLE, we constructed Fam210b knockout (Fam210b^−/−) mice using the CRISPR-Cas9 method. We found that approximately 15.68% of Fam210b^−/− mice spontaneously developed lupus-like autoimmunity, which was characterized by skin ulcerations, splenomegaly, and an increase in anti-double-stranded DNA (anti-dsDNA) IgG antibodies and anti-nuclear antibodies(ANA). Single-cell sequencing showed that Fam210b was mainly expressed in erythroid cells. Critically, the knockout of Fam210b resulted in abnormal erythrocyte differentiation and development in the spleens of mice. Concurrently, the spleens exhibited an increased number of CD71⁺ erythroid cells, along with elevated levels of reactive oxygen species (ROS) in the erythrocytes. The co-culture of CD71⁺ erythroid cells and lymphocytes resulted in lymphocyte activation and promoted dsDNA and IgG production. In summary, Fam210b knockout leads to a low probability of lupus-like symptoms in mice through the overproduction of ROS in CD71⁺ erythroid cells. Thus, Fam210b reduction may serve as a novel key marker that triggers the development of SLE. Full article

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social interaction and restricted and repetitive behaviors. Oxidative stress may be a critical link between mitochondrial dysfunction and ASD as reactive oxygen species (ROS) generated from pro-oxidant environmental toxicants and activated immune cells can result in mitochondrial failure. Recently, mitochondrial dysfunction, autoimmunity, and abnormal lipid mediators have been identified in multiple investigations as an acknowledged etiological mechanism of ASD that can be targeted for therapeutic intervention. Methods: The relationship between lipid mediator markers linked to inflammation induction, such as phospholipase A2/cyclooxygenase-2 (PLA2/Cox-2), and the mitochondrial dysfunction marker anti-mitochondrial antibodies (AMA-M2), and anti-histone autoantibodies in the etiology of ASD was investigated in this study using combined receiver operating characteristic (ROC) curve analyses. This study also sought to identify the linear combination for a given set of markers that optimizes the partial area under ROC curves. This study included 40 age- and sex-matched controls and 40 ASD youngsters. The plasma of both groups was tested for PLA2/COX-2, AMA-M2, and anti-histone autoantibodies’ levels using ELISA kits. ROC curves and logistic regression models were used in the statistical analysis. Results: Using the integrated ROC curve analysis, a notable rise in the area under the curve was noticed. Additionally, the combined markers had markedly improved specificity and sensitivity. Conclusions: The current study suggested that measuring the predictive value of selected biomarkers related to mitochondrial dysfunction, autoimmunity, and lipid metabolism in children with ASD using a ROC curve analysis could lead to a better understanding of the etiological mechanism of ASD as well as its relationship with metabolism. Full article

Elevated oxidative stress can play a pivotal role in autoimmune diseases by exacerbating inflammatory responses and tissue damage. In Sjögren’s disease (SjD), the contribution of oxidative stress in the disease pathogenesis remains unclear. To address this question, we created mice with a tamoxifen-inducible conditional knockout (KO) of a critical antioxidant enzyme, superoxide dismutase 2 (Sod2), in the salivary glands (i-sg-Sod2 KO mice). Following tamoxifen treatment, Sod2 deletion occurred primarily in the ductal epithelium, and the salivary glands showed a significant downregulation of Sod2 expression. At twelve weeks post-treatment, salivary glands from the i-sg-Sod2 KO mice exhibited increased 3-Nitrotyrosine staining. Bulk RNA-seq revealed alterations in gene expression pathways related to ribosome biogenesis, mitochondrial function, and oxidative phosphorylation. Significant changes were noted in genes characteristic of salivary gland ionocytes. The i-sg-Sod2 KO mice developed reversible glandular hypofunction. However, this functional loss was not accompanied by glandular lymphocytic foci or circulating anti-nuclear antibodies. These data demonstrate that although localized oxidative stress in salivary gland ductal cells was insufficient for SjD development, it induced glandular dysfunction. The i-sg-Sod2 KO mouse resembles patients classified as non-Sjögren’s sicca and will be a valuable model for deciphering oxidative-stress-mediated glandular dysfunction and recovery mechanisms. Full article