In this study, six novel iminocoumarin derivatives (5a-5f) were synthesized at room temperature in an acetic medium and structurally characterized using 1H and 13C NMR, FT-IR, mass spectrometry, and elemental analysis. All compounds were obtained in high purity and exhibited spectroscopic features consistent with their proposed structures. In vitro biological evaluation demonstrated significant antitumor activity against A549 non-small cell lung cancer cells, with compound 5e showing potency comparable to carboplatin (IC50 = 28.38 +/- 4.22 & micro;M vs 25.48 +/- 5.22 & micro;M). Additionally, all derivatives effectively inhibited NLRP3 inflammasome activation. Ex vivo assays further confirmed these findings, with compound 5f exhibiting the strongest inhibition, resulting in substantial reductions in IL-1 beta secretion and nitric oxide production. Molecular docking studies revealed strong binding affinities of compound 5e toward EGFR and PD-L1, and of compound 5f toward NLRP3. ADMET predictions indicated favorable pharmacokinetic profiles and low toxicity risk. Overall, these results highlight iminocoumarins, particularly compounds 5e and 5f, as promising multitarget agents with dual anticancer and anti-inflammatory potential, warranting further pharmaceutical investigation.

Radiation therapy is a widely used and effective treatment for various types of cancer; however, it is associated with several side effects, including an increased risk of cardiovascular disease. While the molecular mechanisms underlying RT-induced cardiovascular damage remain unclear, the NLRP3 inflammasome has emerged as a central player in its pathogenesis. This review focuses on the current understanding of the involvement of NLRP3 in radiation-induced cardiotoxicity, highlighting emerging evidence reported in the literature. Recent findings shown that the NLRP3 inflammasome may play a role in radiation-induced cardiovascular diseases, drawing attention to it as a potential therapeutic target. We summarize key findings on the role of radiation in modulating the NLRP3 inflammasome and inducing cardiovascular disease, providing a detailed overview of NLRP3 inflammasome functions in different radiation-induced cardiovascular diseases. Moreover, we discuss the therapeutic potential of targeting NLRP3 to prevent radiation-induced cardiovascular diseases. The development of effective treatments for radiotherapy-induced cardiotoxicity is becoming increasingly urgent and understanding NLRP3-dependent mechanisms could pave the way for innovative therapeutic approaches.

Migraine is a common disorder that affects more than a billion of people worldwide, with an incidence in women three times higher than in men. The medical need for new therapeutic treatments is only partially satisfied by innovative approaches, and a significant percentage of migraineurs are still not satisfied with their therapy. Several lines of evidence links migraine to the activation of the NLRP3-IL-1β pathway. The aim of this research was to test whether NLRP3 blockers may represent innovative therapeutics for migraine. A well-established murine migraine model triggered by nitroglycerine (GTN) was used, together with NLRP3 knockout (Nlrp3(-/-)) mice and the NLRP3 inhibitor MCC950. Both genetic and pharmacological blockade of NLRP3 were effective in reducing and preventing the onset of the periorbital mechanical allodynia (PMA) evoked by GTN in female mice, but not in males. The chronic administration of MCC950 was sufficient to induce PMA on its on, suggesting that the chronic treatment with a NLRP3 blocker may induce medication overuse headache as a side effect. Western blot analysis showed the activation of the NLRP3 pathway in the hippocampus of female mice treated with GTN, but not in their trigeminal ganglion, trigeminal nucleus caudalis and cortex. No activation of the NLRP3 pathway was detected in male mice. Collectively, this study strengthens the view that NLRP3 inhibitors may represent innovative migraine treatments. However, at the same time, it underlines for the first time some limitations that may affect NLRP3 inhibitors, namely sexual dimorphism in drug responsiveness and issues related to their chronic use.

Triple-negative breast cancer (TNBC) is a particularly aggressive and metastatic subtype, characterized by the absence of estrogen, progesterone, and human epidermal growth factor 2 receptors. Outcomes for TNBC patients vary widely, suggesting this classification encompasses different cancers with distinct histological, genomic, and immunological profiles, leading to variable prognoses. The tumor microenvironment, particularly the expression and localization of promyelocytic leukemia protein (PML) in tumor-associated macrophages (TAMs), can influence patient outcomes by modulating inflammation. The beneficial prognostic role of increased tumor-infiltrating lymphocytes (TILs) in TNBC is well-established. In this retrospective study, we found that PML expression in tumor cells is inversely related to the presence of TILs and is associated with poorer outcomes. Patients with disease recurrence exhibited higher levels of TAMs with predominantly nuclear-localized PML, in contrast to patients who showed complete recovery. The accumulation of PML in the nucleus reduces its presence at ER-mitochondria contact sites, impairing its interaction with the NLRP3 inflammasome and leading to increased IL-1β secretion. This promotes a pro-inflammatory tumor microenvironment as seen in patients with adverse outcomes. Our findings suggest that both PML expression in cancer cells and its localization in TAMs can serve as additional prognostic factors, highlighting the potential of PML as a therapeutic target in TNBC.

Fusobacterium nucleatum (F. nucleatum), a Gram-negative anaerobe, is increasingly implicated in the pathogenesis of colorectal cancer (CRC) and inflammatory bowel disease (IBD). Its adhesin FadA enables epithelial adherence and invasion, promoting inflammation and tumorigenesis. F. nucleatum has been shown to activate the NLRP3 inflammasome, leading to IL-1β release, and is associated with chemoresistance and poor prognosis in CRC. Additionally, lipid peroxidation markers such as malondialdehyde (MDA) and 4-hydroxy-nonenal (4-HNA) may contribute to inflammation-driven carcinogenesis. This study protocol aims to investigate the role of F. nucleatum in the development and progression of IBD and CRC through integrated clinical, molecular, and imaging approaches. The protocol involves quantifying F. nucleatum in tissue biopsies across disease stages and assessing correlations with inflammatory and oxidative markers. It will explore the bacterium’s involvement in NLRP3 inflammasome activation, IL-1β production, and autophagy, and its potential contribution to chemoresistance. Furthermore, radiomic analysis of computed tomography (CT) images will be performed to identify imaging phenotypes associated with microbial load and inflammatory activity. Although primarily a protocol, the study includes preliminary in vitro data showing that exposure to FadA significantly increases inflammatory markers in Caco-2 cells, supporting the hypothesis that F. nucleatum contributes to a pro-inflammatory, pro-tumorigenic microenvironment relevant to CRC progression.

The integrin effector, PTRH2, associates with mitochondria in adherent cells where its function has not been elucidated (Jan Y, et al. 2004. A mitochondrial protein, Bit1, mediates apoptosis regulated by integrins and Groucho/TLE corepressors. Cell. 116:751-762; Griffiths GS, et al. 2011. Bit-1 mediates integrin-dependent cell survival through activation of the NF{kappa}B pathway. J Biol Chem. 286:14713-14723). PTRH2 loss-of-function mutations cause multisystem disease in children through an unknown mechanism. We sought to determine the role of mitochondrial PTRH2. We used immunoprecipitation/mass spectrometric proteomics to identify PTRH2 interacting partners: TRABID (a deubiquitinase [DUB]) and respiratory complex I NADH: ubiquinone oxidoreductase core subunit 5 (mt-ND5). We show for the first time that mitochondrial PTRH2 regulates TRABID's ability to deubiquitylate mt-ND5. In cells lacking PTRH2 expression, mt-ND5 stability is significantly increased due to aberrant TRABID-mediated deubiquitylation of mt-ND5. This increase in mt-ND5 stability promotes complex I activity and ATP production, which under stress conditions leads to mitochondrial Ca2+ overload. Reexpression of mitochondrial PTRH2 blocks TRABID-mediated mt-ND5 deubiquitylation, resulting in mt-ND5 polyubiquitylation and proteasomal degradation. Inhibiting complex I or TRABID activity rescued PTRH2 loss-of-function mutant patient cells from mitochondrial Ca2+ overload under stress. Immunostaining analysis of ptrh2+/+ and ptrh2-/- mouse skeletal muscle revealed a negative relationship between PTRH2 and mt-ND5, confirming a regulatory role for PTRH2 in controlling mt-ND5 stability. Taken together, mitochondrial PTRH2 is a regulator of metabolic homeostasis that, when lost, promotes mitochondrial Ca2+ overload when cells are exposed to stress signals. Targeting mt-ND5 stability through PTRH2-mediated regulation of TRABID's DUB function provides a novel mechanistic approach to inhibit mitochondrial Ca2+ overload in diseases that occur due to dysregulated mitochondria.

Objectives: Despite optimal local control obtained with neoadjuvant chemoradiotherapy (CRT), data on overall survival (OS) and disease-free survival (DFS) of local advanced rectal cancer patients are still equivocal. This meta-analysis aimed to estimate the pathological complete response (pCR), regression rate, DFS, and OS probabilities of rectal cancer patients treated with a second chemotherapy drug added to fluoropyrimidine and long-term radiotherapy. Methods: Computerized bibliographic searches of MEDLINE, PUBMED, Web of Science and the Cochrane Central Register of Controlled Trials databases (1970–2023) were supplemented with hand searches of reference lists. Studies were included if they were randomised controlled trials (RCTs) comparing intensified chemotherapy with CRT to preoperative CRT and if they had patients with resectable, histologically proven rectal adenocarcinoma without metastases. Results: Eighteen RCTs (7695 patients) were analysed. Data on population, intervention, and outcomes were extracted from each RCT, following the intention-to-treat method, by three independent observers and combined using the DerSimonian and Laird methods. A chemotherapy with two drug and long-term radiotherapy CRT, compared to preoperative CRT (fluoropyrimidine and long-term radiotherapy), significantly increases the rate of pathological complete response (OR 1.37 (95% CI, 1.16–1.63) p = 0.0003) and the regression rate (OR 1.57 (95% CI, 1.16–2.14) p < 0.00001). Furthermore, it increases DFS (HR 0.87 (95% CI, 0.79 to 0.95) p = 0.002 and OS HR 0.84 (95% CI, 0.74 to 0.95) p = 0.007). The risk of severe adverse events (≥G3) is increased OR 1.96 (95% CI 1.35–2.85), p = 0.0005. Conclusions: In patients with resectable rectal cancer, intensified chemotherapy can reduce by 13% the risk of disease progression and by 16% the risk of death.

p66Shc is an adaptor protein and one of the cellular fate regulators since it modulates mitogenic signaling pathways, mitochondrial function, and reactive oxygen species (ROS) production. p66Shc is localized mostly in the cytosol and endoplasmic reticulum (ER); however, under oxidative stress, p66Shc is post-translationally modified and relocates to mitochondria. p66Shc was found in the intermembrane space, where it interacts with cytochrome c, contributing to the hydrogen peroxide generation by the mitochondrial respiratory chain. Our previous studies suggested that p66Shc is localized also in mitochondria-associated membranes (MAM). MAM fraction consists of mitochondria and mostly ER membranes. Contact sites between ER and mitochondria host proteins involved in multiple processes including calcium homeostasis, apoptosis, and autophagy regulation. Thus, p66Shc in MAM could participate in processes related to cell fate determination. Due to reports on various and conditional p66Shc intracellular localization, in the present paper, we describe the allocation of p66Shc pools in different subcellular compartments in mouse liver tissue and HepG2 cell culture. We provide additional evidence for p66Shc localization in MAM. In the present study, we use precisely purified subcellular fraction isolated by differential centrifugation-based protocol from control mouse liver tissue and HepG2 cells and from cells treated with hydrogen peroxide to promote mitochondrial p66Shc translocation. We performed controlled digestion of crude mitochondrial fraction, in which the degradation patterns of p66Shc and MAM fraction marker proteins were comparable. Moreover, we assessed the distribution of the individual ShcA isoforms (p46Shc, p52Shc, and p66Shc) in the subcellular fractions and their contribution to the total ShcA in control mice livers and HepG2 cells. In conclusion, we showed that a substantial pool of p66Shc protein resides in MAM in control conditions and after oxidative stress induction.

Background Triple-negative breast cancer (TNBC) stands out as a particularly aggressive and metastatic subtype, defined by its absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. It's important to note that outcomes for TNBC patients vary widely, suggesting this classification encompasses various cancers with different histological, genomic, and immunological profiles, leading to distinct prognoses. Our prior research revealed that the tumor microenvironment, especially the expression and localization of promyelocytic leukemia protein (PML) within tumor-associated macrophages (TAMs), can influence patient outcomes by affecting the inflammatory environment. The beneficial prognostic role of increased tumor-infiltrating lymphocytes (TILs) in TNBC is also well-established. Methods This is a retrospective cohort study composed of a total of 20 female patients affected by TNBCS, divided into two groups according to the observed outcome in a 10-year follow-up: 10 TNBC patients showing complete remission of the disease and 10 TNBC patients experiencing tumor recurrence and death within the follow-up. The collected histological tumor samples were stained to evaluate TILs and TAMs presence and PML expression. Results We found that PML expression in tumor cells is inversely related to the presence of TILs and is associated with worse patient outcomes. Furthermore, we discovered that patients with disease recurrence exhibited a higher number of TAMs with predominantly nuclear-localized PML, unlike what was observed in patients with complete disease recovery. This accumulation of PML in the nucleus reduces its presence at ER-mitochondria contact sites, thereby impairing its interaction with the NLRP3 inflammasome and leading to increased IL-1β secretion. This promotes a pro-inflammatory tumor microenvironment, as seen in patients with adverse outcomes. Conclusions Our findings suggest that both PML expression in cancer cells and its localization in TAMs can serve as additional prognostic factors, alongside TIL abundance, indicating the potential of PML as a target for TNBC therapy.

Methicillin-resistant Staphylococcus aureus (MRSA) is the most common causative agent of acute bacterial skin and skin-structure infections (ABSSSI), one of the major challenges to the health system worldwide. Although the use of antibiotics as the first line of intervention for MRSA-infected wounds is recommended, important side effects could occur, including cytotoxicity or immune dysregulation, thus affecting the repair process. Here, we show that the oxazolidinone antibiotic linezolid (LZD) impairs wound healing by aberrantly increasing interleukin 1 β (IL-1β) production in keratinocytes. Mechanistically, LZD triggers a reactive oxygen species (ROS)-independent mitochondrial damage that culminates in increased tethering between the endoplasmic reticulum (ER) and mitochondria, which in turn activates the NLR family pyrin domain-containing 3 (NLRP3) inflammasome complex by promoting its assembly to the mitochondrial surface. Downregulation of ER-mitochondria contact formation is sufficient to inhibit the LZD-driven NLRP3 inflammasome activation and IL-1β production, restoring wound closure. These results identify the ER-mitochondria association as a key factor for NLRP3 activation and reveal a new mechanism in the regulation of the wound healing process that might be clinically relevant.

Mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) emerged to play critical roles in numerous cellular processes, and their dysregulation has been associated to neurodegenerative disorders. Mutations in the SPG4 gene coding for spastin are among the main causes of hereditary spastic paraplegia (HSP). Spastin binds and severs microtubules, and the long isoform of this protein, namely M1, spans the outer leaflet of ER membrane where it interacts with other ER-HSP proteins. Here, we showed that overexpressed M1 spastin localizes in ER-mitochondria intersections and that endogenous spastin accumulates in MERCs. We demonstrated in different cellular models that downregulation of spastin enhances the number of MERCs, alters mitochondrial morphology, and impairs ER and mitochondrial calcium homeostasis. These effects are associated with reduced mitochondrial membrane potential, oxygen species levels, and oxidative metabolism. These findings extend our knowledge on the role of spastin in the ER and suggest MERCs deregulation as potential causes of SPG4-HSP disease.

Objectives: To evaluate the in vitro effect of tofacitinib on autophagy activity of psoriatic arthritis (PsA) fibroblastlike synoviocytes (FLS), and to confirm its activity on infiammatory and invasive properties of FLS and synovial cells, deepening the impact on mitochondrial function.Methods: FLS, peripheral blood mononuclear cells (PBMCs), and synovial cells from active PsA patients were cultured with tofacitinib 1 mu M or vehicle control for 24 h. Autophagy was measured by Western blot and by fiuorescence microscopy. Chemokines/cytokines released into culture supernatants were quantified by ELISA, while invasive properties of FLS by migration assays. Specific mitochondrial probes were adopted to measure intracellular reactive oxygen species (ROS), mitochondrial potential, morphology, turnover and mitophagy. Oxygen consumption rate (OCR), refiecting oxidative phosphorylation, was quantified using the Seahorse technology. Differences were determined by adopting the non-parametric Wilcoxon signed rank test.Results: 18 patients with moderately-to-severely active PsA were enrolled. Tofacitinib significantly increased the levels of the autophagy markers LC3-II and ATG7 in PsA FLS compared to vehicle control, suggesting an increase in spontaneous autophagy activity; no effect was highlighted in PBMCs and synovial cells cultures. Tofacitinib reduced migration properties of PsA FLS, and reduced MCP-1 and IL-6 release into FLS and synovial cells cultures supernatants. Furthermore, tofacitinib decreased intracellular ROS production, increased basal OCR, ATP production and maximal respiratory capacity, and enhanced mitophagy and mitochondrial turnover.Conclusions: The JAK inhibitor tofacitinib reduces the pro-invasive and pro-infiammatory properties of PsA FLS. Autophagy induction and mitochondrial quality control modulation by tofacitinib might contribute to FLS function restoration.

The aims of this systematic literature review (SLR) were to identify the effects of approved biological and targeted synthetic disease modifying antirheumatic drugs (b/tsDMARDs) on synovial membrane of psoriatic arthritis (PsA) patients, and to determine the existence of histological/molecular biomarkers of response to therapy. A search was conducted on MEDLINE, Embase, Scopus, and Cochrane Library (PROSPERO:CRD42022304986) to retrieve data on longitudinal change of biomarkers in paired synovial biopsies and in vitro studies. A meta-analysis was conducted by adopting the standardized mean difference (SMD) as a measure of the effect. Twenty-two studies were included (19 longitudinal, 3 in vitro). In longitudinal studies, TNF inhibitors were the most used drugs, while, for in vitro studies, JAK inhibitors or adalimumab/secukinumab were assessed. The main technique used was immunohistochemistry (longitudinal studies). The meta-analysis showed a significant reduction in both CD3+ lymphocytes (SMD -0.85 [95% CI -1.23; -0.47]) and CD68+ macrophages (sublining, sl) (SMD -0.74 [-1.16; -0.32]) in synovial biopsies from patients treated for 4-12 weeks with bDMARDs. Reduction in CD3+ mostly correlated with clinical response. Despite heterogeneity among the biomarkers evaluated, the reduction in CD3+/CD68+sl cells during the first 3 months of treatment with TNF inhibitors represents the most consistent variation reported in the literature.

Substrate degradation by the ubiquitin proteasome system (UPS) in specific membrane compartments remains elusive. Here, we show that the interplay of two lipid modifications and PDE6δ regulates compartmental substrate targeting via the SCFFBXL2. FBXL2 is palmitoylated in a prenylation-dependent manner on cysteines 417 and 419 juxtaposed to the CaaX motif. Palmitoylation/depalmitoylation regulates its subcellular trafficking for substrate engagement and degradation. To control its subcellular distribution, lipid-modified FBXL2 interacts with PDE6δ. Perturbing the equilibrium between FBXL2 and PDE6δ disrupts the delivery of FBXL2 to all membrane compartments, whereas depalmitoylated FBXL2 is enriched on the endoplasmic reticulum (ER). Depalmitoylated FBXL2(C417S/C419S) promotes the degradation of IP3R3 at the ER, inhibits IP3R3-dependent mitochondrial calcium overload, and counteracts calcium-dependent cell death upon oxidative stress. In contrast, disrupting the PDE6δ-FBXL2 equilibrium has the opposite effect. These findings describe a mechanism underlying spatially-restricted substrate degradation and suggest that inhibition of FBXL2 palmitoylation and/or binding to PDE6δ may offer therapeutic benefits.

Neuroinflammation represents a dynamic process of defense and protection against the harmful action of infectious agents or other detrimental stimuli in the central nervous system (CNS). However, the uncontrolled regulation of this physiological process is strongly associated with serious dysfunctional neuronal issues linked to the progression of CNS disorders. Moreover, it has been widely demonstrated that neuroinflammation is linked to epilepsy, one of the most prevalent and serious brain disorders worldwide. Indeed, NLRP3, one of the most well-studied inflammasomes, is involved in the generation of epileptic seizures, events that characterize this pathological condition. In this context, several pieces of evidence have shown that the NLRP3 inflammasome plays a central role in the pathophysiology of mesial temporal lobe epilepsy (mTLE). Based on an extensive review of the literature on the role of NLRP3-dependent inflammation in epilepsy, in this review we discuss our current understanding of the connection between NLRP3 inflammasome activation and progressive neurodegeneration in epilepsy. The goal of the review is to cover as many of the various known epilepsy models as possible, providing a broad overview of the current literature. Lastly, we also propose some of the present therapeutic strategies targeting NLRP3, aiming to provide potential insights for future studies.

Abstract Background & Aims: Despite optimal local control, chemoradiotherapy (CRT) data on overall and disease-free survival are still equivocal. This meta-analysis aimed to estimate the pathological complete response (pCR), regression rate, disease-free survival and overall survival probabilities of rectal cancer patients treated with intensified chemotherapy. Data sources: Computerised bibliographic searches of MEDLINE and Cochrane Central Register of Controlled Trials databases (1970-2023) were supplemented with hand searches of reference lists. Study selection: Studies were included if they were randomised controlled trials (RCTs) comparing intensified chemotherapy with CRT to preoperative CRT and if they had patients with resectable, histologically-proven, rectal adenocarcinoma without metastases. Eighteen RCTs (7695 patients) were analysed. Data extraction: Data on population, intervention, and outcomes were extracted from each RCT, following the intention-to-treat method, by three independent observers and combined using the DerSimonian and Laird methods. Results: Intensified chemotherapy and CRT, compared to preoperative CRT, significantly increases the rate of pathological complete response (OR 1.37 (95% CI, 1.16-1.63) p=0.0003) and the regression rate (OR 1.57 (95% CI, 1.16-2.14) p < 0.00001). Furthermore, it increases disease-free survival HR 0.87 (95% CI, 0.79 to 0.95) p=0.002 and overall survival HR 0.84 (95% CI, 0.74 to 0.95) p= 0.007. Finally, the risk of therapy to severe adverse events (≥G3) is increased OR 1.96 (95% CI 1.35–2.85), p =0.0005. Conclusions: In patients with resectable rectal cancer, intensified chemotherapy can reduce by 13% the hazard of disease progression and by 16% the hazard of death.

The NLRP3 inflammasome is a critical component of innate immunity that senses diverse pathogen- and host-derived molecules. However, its aberrant activation has been associated with the pathogenesis of multiple diseases, including cancer. In this study, we designed and synthesized a series of aryl sulfonamide derivatives (ASDs) to inhibit the NLRP3 inflammasome. Among these, compounds 6c, 7n, and 10 specifically inhibited NLRP3 activation at nanomolar concentrations without affecting the activation of the NLRC4 and AIM2 inflammasomes. Furthermore, we demonstrated that these compounds reduce interleukin-1β (IL-1β) production in vivo and attenuate melanoma tumor growth. Moreover, metabolic stability in liver microsomes of 6c, 7n, and 10 was studied along with plasma exposure in mice of the most interesting compound 6c. Therefore, we generated potent NLRP3 inflammasome inhibitors, which can be considered in future medicinal chemistry and pharmacological studies aimed at developing a new therapeutic approach for NLRP3 inflammasome-driven cancer.

Uncontrolled inflammatory response arising from the tumor microenvironment (TME) significantly contributes to cancer progression, prompting an investigation and careful evaluation of counter-regulatory mechanisms. We identified a trimeric complex at the mitochondria-associated membranes (MAMs), in which the purinergic P2X7 receptor - NLRP3 inflammasome liaison is fine-tuned by the tumor suppressor PML. PML downregulation drives an exacerbated immune response due to a loss of P2X7R-NLRP3 restraint that boosts tumor growth. PML mislocalization from MAMs elicits an uncontrolled NLRP3 activation, and consequent cytokines blast fueling cancer and worsening the tumor prognosis in different human cancers. New mechanistic insights are provided for the PML-P2X7R-NLRP3 axis to govern the TME in human carcinogenesis, fostering new targeted therapeutic approaches.