Introduction: Cellular senescence is a tumour-suppressive mechanism that contributes to ageing and age-related diseases. Among the strategies developed to eliminate senescent cells (SCs), senolytic drugs are promising, although their mechanisms of action remain to be fully elucidated. In this study, we investigated the senolytic effect of quercetin on doxorubicin (Doxo)-induced senescent fibroblasts, exploring a novel underlying mechanism and focusing on its ability to reduce the pro-tumourigenic impact of these cells on osteosarcoma cells (U2OS).

Methods: Doxo-induced senescent WI-38 fibroblasts were treated with quercetin, alone or in combination with the mTOR inhibitor PP242 or the ER stress inhibitor 4-PBA. Senescence markers, intracellular calcium (Fluo-4-AM), and cell death (Annexin V/7-AAD) were evaluated. Autophagy was assessed via western blot and immunofluorescence (Beclin-1, LC3-I/II, p62), while ER stress was monitored through the detection of spliced XBP1 mRNA. The pro-tumour effects of SCs were evaluated using U2OS colony formation and invasion assays with conditioned media (CM).

Results: Senescent fibroblasts displayed high autophagy, likely as a compensatory response to ER stress. Quercetin selectively eliminated ~30% of SCs by suppressing autophagy, thereby exacerbating ER stress and inducing cell death. This effect was abolished by co-treatment with PP242 or 4-PBA, confirming the role of autophagy and ER stress in quercetin’s senolytic action. Moreover, quercetin-treated SCs produced CM that significantly reduced the aggressiveness of U2OS cells.

Conclusion: Our findings reveal a novel mechanism of quercetin-induced senolysis involving autophagy inhibition and ER stress and demonstrate that even partial clearance of SCs can mitigate their tumour-promoting effects.

A growing body of evidence suggests that interventions targeting fundamental mechanisms of biological aging may represent effective strategies to promote healthy aging and delay the onset of age-related conditions such as frailty. Among these, the use of senolytic compounds—capable of selectively clearing senescent cells—and microbiome modulators like probiotics has shown promising results in preclinical models. However, the potential synergistic effects of combining these two strategies remain largely unexplored.

In this study, we investigate the impact of a combinatorial treatment using a novel senolytic agent and a probiotic formulation enriched in Akkermansia muciniphila, a bacterium previously associated with improved gut barrier integrity and reduced inflammaging. Both compounds, when used individually, have shown beneficial effects on lifespan and healthspan in preclinical studies. Here, we tested their synergistic potential in naturally aged mice through a longitudinal experimental design.

Carnitine is considered a vitamin-like molecule because up to 75% of the body's pool in humans is sourced from the diet, as the evolutionary loss of a crucial enzyme in the biosynthetic pathway limits its production. The vital carnitine role is due to its requirement by the mitochondrial fatty acid β-oxidation, as it has been well acknowledged for decades. In the SASP context and age-related conditions, carnitine was revealed to be important not only for energetics but also for anti-oxidant and anti-inflammatory effects; moreover, carnitine and its acetyl derivative are often used as dietary supplements for treating neurological disorders. Therefore, it is not trivial that, besides enzyme-handling carnitine, pivotal roles are played by membrane transporters responsible for carnitine and acetyl-carnitine traffic among tissues (s) and, within a cell, among compartments. During the AGE-IT program, two SLC transporters, crucial for the carnitine network, have been investigated by employing a multidisciplinary approach of ex-vivo, in silico and in vitro tools: the plasma membrane transporter SLC22A4 (OCTN1) and the mitochondrial transporter SLC25A20 (CAC). OCTN1 belongs to the SLC22 family, including membrane transporters of cations, anions and zwitterions. We investigated the transport mechanism of carnitine via OCTN1, following the identification of an OCTN1 SNP involved in frailty. Furthermore, we have described the interaction between the CAC, i.e., the only route for acyl-carnitine entry in mitochondria, and itaconate, an endogenous compound produced during inflammation. We described the formation of the covalent interaction of itaconate with a Cys residue of CAC with consequent modulation of the CAC activity. These results shed new light on the role of carnitine transporters in inflammation, opening perspectives for further development of senolytics.

Hypoxia and ageing share a common feature: impaired oxygen delivery to tissues. This condition is known to compromise endothelial cell (EC) function and to promote senescence, particularly in the presence of additional pathological stimuli such as chronic hyperglycaemia, inflammation, and oxidative stress.

We assessed gene expression and protein secretion levels of key inflammatory mediators, markers of oxidative stress, and hallmarks of cellular senescence in ECs exposed to high glucose (33 mM for 72 hours under either normoxic conditions or prolonged hypoxia (16 hours), followed by 2 hours of reoxygenation.

ECs exposed to high glucose and hypoxia developed a senescent phenotype, as indicated by increased expression of p21 and p16, and elevated β-galactosidase staining. Surprisingly, this was not associated with the classical senescence-associated secretory phenotype (SASP). Instead, ECs showed selective upregulation of angiogenic factors VEGF and HB-EGF. Compared to normoxia, ECs exposed to hypoxia, especially in the presence of high glucose, exhibited reduced transcription and secretion of NF-κB-dependent cytokines and chemokines (MCP-1, IL-6, IL-8, and TRAIL), along with downregulation of NF-κB-p50. Conversely, we observed a strong upregulation of A20 and TNIP3, two well-characterised negative regulators of NF-κB signalling. Senescence induced by hypoxia and hyperglycaemia does not trigger a typical inflammatory SASP. Instead, ECs activate an anti-inflammatory response, suppressing NF-κB signalling and enhancing the expression of its inhibitors A20 and TNIP3. Although they enter a senescent state, ECs activate a compensatory anti-inflammatory and cytoprotective response. This involves upregulating stress-adaptive pathways that preserve endothelial function, limit oxidative damage and protect nearby cells.

The senescence-associated secretory phenotype (SASP) characterizes senescent cells and contributes to inflammaging, one of the many aging-associated dysfunctions.

SASP induces extracellular matrix remodeling and promotes a hyperinflammatory state in the organism, thanks to the secretion of several interleukins, chemokines and matrix metalloproteases, which act both in an autocrine and paracrine manner to induce senescence.

However, the mechanisms of SASP that associate the altered synthesis, secretion and distribution of SASP components in the cellular microenvironment with the aging process are poorly understood. We aim to investigate how the secretory pathway in senescent cells determines the post-translational regulation of SASP proteins, focusing on protein sorting and export at the trans-Golgi network (TGN). Our preliminary data have highlighted the presence of a sophisticated signaling network in the secretory pathway that regulates different classes of proteins. Thanks to cell type-specific components, different proteins can activate distinct regulatory mechanisms localized in the TGN to be correctly transported and secreted. As a fundamental basis for subsequent studies, we set up and conducted imaging-based studies to investigate the intracellular trafficking and secretion of a panel of reporter proteins (with different sorting characteristics, such as basolateral, apical and lysosomal), in a set of model cell lines, to identify their specific requirements for TGN exit. Our goal is to identify the regulatory mechanisms controlling the secretion of the main SASP components that may contribute to the identification of biomarkers of aging and define pharmacological strategies able to modulate and normalize SASP secretion as a novel approach for anti-aging therapy.

Cellular senescence is defined by a stable arrest of cell division even under optimal growth conditions. The accumulation of senescent cells in aging tissues contributes to various age-related pathological conditions. Here, we show that the Golgi complex, a key organelle involved in protein processing and secretion, undergoes structural alterations in senescent cells. By treating cultured cells with genotoxic agents, such as doxorubicin and etoposide, we observed significant morphological changes in the Golgi apparatus and modification of Golgi proteins. Focusing on GRASP65, a structural protein essential for maintaining Golgi ribbon integrity, we found that genotoxic stress led to its phosphorylation, resulting in partial Golgi disassembly. This fragmentation correlates with the increased expression of classical senescence markers. Importantly, treatment with SP600125, an inhibitor of JNK2-mediated GRASP65 phosphorylation, preserved Golgi ribbon continuity and significantly reduced senescence marker expression. Furthermore, we designed a GRASP65-mimetic peptide that is capable of reversing Golgi disassembly. Our findings suggest that the development of a strategy to restore Golgi integrity can induce a decrease of senescent cells and inactivate the senescence-associated secretory phenotype (SASP), which has a pro-inflammatory role in the promotion of tumor growth.

Frailty and systemic inflammation are key features of biological aging, but their combined impact on mortality in older adults remains unclear. This study aimed to evaluate the association of frailty and circulating inflammatory biomarkers with long-term mortality in a hospitalized geriatric population. We analyzed data from 1,009 patients (median age 84) enrolled in the ReportAGE study across three Italian geriatric hospitals. Frailty was assessed using a deficit accumulation-based Frailty Index (FI), and serum levels of IL-6, IL-10, and CXCL9 were measured by immunoassay. A composite inflammaging score (I3 score) was calculated by summing risk tiers assigned to each biomarker. Associations with 10-year all-cause mortality were evaluated using Cox regression models adjusted for clinical and laboratory variables. Our results confirmed the I3 scores were independently associated with increased mortality risk (HR = 2.42, high vs. low), with CXCL9 emerging as the strongest individual predictor. Notably, the I3 score provided additional prognostic value beyond FI alone. Combined stratification by FI and I3 identified four distinct mortality risk groups, with the highest risk observed in patients with both high frailty and high inflammation. These findings highlight the value of integrating inflammaging biomarkers and frailty into risk assessment models, offering clinically relevant tools to improve prognostication in older adults.