Ferroptosis recently gained considerable attention in breast cancer research, particularly in triple-negative subtypes. In this context, emerging evidence links transglutaminase 2 (TG2) to aggressive tumour phenotypes, highlighting pharmacological TG2 inhibition as a promising therapeutic approach in aggressive breast cancer settings.Comparative analysis of independent RNA-sequencing datasets from distinct cellular models treated with irreversible TG2 inhibitors revealed ferroptosis as the only significantly enriched and commonly deregulated process. In triple-negative breast cancer cells, treatment with the cell-permeable AA9 inhibitor triggered a coherent ferroptotic transcriptional program, characterized by downregulation of glutathione peroxidase 4 and the glutathione biosynthesis genes and by upregulation of stress-responsive and iron-handling factors. A covalent pull-down magnetic system combined with proteomics and bioinformatic target prediction identified TG2 as the primary intracellular target of AA9 and revealed a TG2-centered interactome involving, among the pathways, hypoxia signalling, cytoskeletal regulation, and apoptosis. Moreover, molecular docking and immunoprecipitation experiments demonstrated that TG2 is physically associated with BH3-interacting domain death agonist and Bcl-2-associated X protein, key mediators of ferroptosis and apoptosis. Transmission electron microscopy further confirmed that AA9 induced ferroptosis-associated mitochondrial alterations, prevented by ferrostatin-1 co-treatment.Overall, these findings confirm TG2 pharmacological targeting as a strategy to reprogram metabolic adaptation and cell death responses in aggressive tumour subtypes.

Background: MiRNAs within extracellular vesicles can encompass body barriers, reflecting stage, progression, and response to treatments of various diseases, including multiple sclerosis (MS)—a chronic immune-mediated disease of the central nervous system that causes progressive disability, with highly variable clinical courses. In this context, urinary exosomal miRNAs could be an appealing source of biomarkers, thanks to their non-invasive and easily repeatable collection. Methods: In this exploratory investigation, we tried to assess if profiling urinary exosomal miRNAs could reveal subtle differences within an apparently homogeneous MS population. The study involved 24 patients with primary or secondary progressive MS, whose urinary exosomes (UEs) were subjected to evaluation of a panel of 87 miRNAs variously correlated with neuroinflammation, cardiovascular functions, and/or involved in MS. Results: We revealed that the examined miRNAs were heterogeneously expressed across the patients, reflecting, as expected, their gender and/or hormonal status. Two miRNAs discriminated against primary or secondary progressive MS, and a panel of 14 commonly upmodulated miRNAs identified patients with longer disease duration and a greater degree of disability. Conclusions: Even if preliminary, these data represent the first relationship between UEs and MS features in humans and suggest that urine could constitute a non-invasive source of exosomal miRNAs, which could prove useful in complementing conventional monitoring to provide a more personalized management of MS patients.

Psoriasis is a chronic, systemic inflammatory disease associated with cardiovascular and metabolic comorbidities. It is driven by a sustained immune response in the skin, orchestrated by keratinocytes and both innate and adaptive immune cells. The initial phase of the pathology remains largely unclear. It likely involves the activation of dendritic cells by stress signals from keratinocytes with the NLRP3 inflammasome potentially acting as a key innate sensor. Here we investigate the effects of the topical administration of the selective NLRP3 inhibitor MCC950, in the imiquimod-induced psoriasis-like inflammation model with the aim to achieve the inhibition of both cutaneous and systemic manifestations. Topical MCC950 effectively suppressed NLRP3 inflammasome activation by preventing pro-caspase-1 cleavage and generation of the active p20 subunit in the skin. Gene expression analysis showed that inhibition of NLRP3 suppressed the expression of psoriasis hallmark genes, including Il17, Tnf, S100a8/a9 and Il1b. Furthermore, network analysis identified NLRP3 as a central hub in the psoriasis-associated inflammatory module, supporting the concept that inflammasome inhibition dampens multiple downstream inflammatory pathways. In psoriasis-like condition, MCC950 reduced the skin infiltration of CD3+ T cells, CD11b+ myeloid and CD11c+ dendritic cells. Notably, MCC950 also prevented the systemic effects of psoriasis-like inflammation. Analysis in the spleen shows that it effectively prevented the phenotypic changes induced by psoriasis-like inflammation including the increased frequency of IL-17 A+ T cells. Finally, MCC950 prevented the systemic increase of serum IL-6. These findings highlight that upstream targeting of the NLRP3 inflammasome by MCC950 represents a promising strategy to inhibit both initiation and the systemic spread of psoriasis-like inflammation.

Human-induced pluripotent stem cells (hiPSCs) have emerged as a promising source of transplantable insulinproducing cells (IPCs) to restore insulin levels in Type 1 Diabetes (T1D) patients. Despite progress, obtaining fully functional β cells from hiPSCs remains challenging, underscoring the need to better understand the intracellular mechanisms involved. We investigated here the potential role of Vav1, a multidomain protein that we identified as crucial for the maturation of human biliary stem cells (hBTSCs) into β-like cells and in the trans-differentiation of pancreatic adenocarcinoma (PDAC) cells into IPCs; Levels and subcellular localization of Vav1 were investigated throughout a seven-step differentiation process of hiPSCs to β cells. Vav1expression was forcedly modulated in pancreatic progenitors, and the potential effects were evaluated on insulin production and on PDX1, miR-375, and Akt, key regulators of β cells generation; RESULTS. Vav1 showed dynamic modulation, with pancreatic precursor cells requiring adequate levels of the protein to generate IPCs. Vav1 sustains the expression of PDX1, a primary regulator of insulin expression, and of its target miR-375, essential for determining β cell mass. Furthermore, Vav1 reduction correlated with increased activation of Akt, which regulates cell survival and insulin secretion in β cells and is down-regulated by miR- 375. Our findings suggest the existence of a Vav1/PDX1/miR-375/Akt axis as part of the complex network orchestrating the generation of functional β cells. These insights indicate that strategies aimed at specifically modulating Vav1 levels may positively impact the generation of IPCs in vitro and, ultimately, β cell replacement therapy for T1D.

We have investigated the involvement of K+ channels in generating the membrane current in MDA-MB-436 cells, a model of triple-negative breast cancer (TNBC). The membrane current is strongly influenced by the opening of voltage-dependent channels insensitive to the nonspecific K+ channel inhibitor 4-aminopyridine (4-AP). Using the cell patch clamp technique, we observed a significant decrease in membrane current after exposure to the generic K+ channel inhibitor tetraethylammonium chloride (TEA-Cl), indicating that K+ ions contribute to the overall membrane current through K+ channels that are insensitive to 4-AP but TEA-Cl-sensitive. RNA-sequencing analysis identified the Big Potassium (BK or Maxi-K or KCa1.1, encoded by KCNMA1) and the Kv2.1 (encoded by KCNB1) channels as putative candidates, both of which are involved in cancer cell proliferation and migration. Iberiotoxin, a specific inhibitor of BK channels, did not affect the total membrane current, just as CdCl₂ did, a potent inhibitor of Ca2⁺ channels involved in BK activation. Using selective inhibitors, stromatoxin and drofenine, we demonstrated that the Kv2.1 channel contributes to the membrane current in MDA-MB-436 cells. Furthermore, drofenine inhibited cell migration as measured by the xCELLigence Real-Time Cell Analyzer System and induced apoptosis. Single-cell analysis revealed that the Kv2.1 channel is expressed in both normal and cancerous tissues, with significant upregulation in brain metastases. This raises the possibility that Kv2.1 could be explored as a potential therapeutic target for controlling advanced stages of the neoplasia.

Diabetes is a growing global health concern, calling for improved diagnostic and therapeutic strategies. Of the emerging possible biomarkers, microRNA 375 (miR-375) has gained attention for its pivotal role in pancreatic β cell development and function, and its altered blood levels following β cell injury. This review summarizes the current knowledge on the role of miR-375 in insulin regulation, its correlation with diabetes, and its clinical potential. Despite its well-known role in β cell biology, literature analyses have failed to reveal a consistent correlation between the circulating levels of miR-375 and diabetes. A key limitation lies in the lack of β cell specificity of miR-375, along with its modulation by diabetes-related complications, which influences circulating levels of the miRNA. Moreover, the absence of large-scale, standardized clinical studies undermines the comparability of existing data. Despite these limits, the literature analysis clearly indicates the need to expand research into miR-375 modulation strategies in humans, as integrating miR-375 with other diagnostic and therapeutic technologies could enhance its clinical relevance. Such strategies may support more personalized and timely interventions for treating diabetes and its complications, ultimately benefiting patient outcomes and contributing to the sustainability of global healthcare systems.

Background/Objectives: Natural compounds are being increasingly explored as potential adjuvants to conventional drugs in oncological treatments. Regarding breast tumors, several studies indicate that garlic (Allium sativum) may protect against onset, counteracts aggressiveness, and prevents malignant progression of cells from non-invasive tumors. It has been widely demonstrated that garlic derivatives induce apoptosis and reduce invasive potential in ER-positive and triple-negative breast tumor cells. However, the current literature lacks studies investigating their effects on HER2-positive (HER2+) breast cancers. This study therefore aimed to explore the effects of a garlic extract and diallyl disulfide (DADS), one of its most bioactive organosulfur compounds, on HER2+ phenotype breast tumor cells. Methods: The effects of a garlic extract and diallyl disulfide (DADS) were investigated on MDA-MB-453 and SKBR3 breast tumor cell lines. Cell growth, invasive potential, and Akt-related signaling were assessed after 4–72 h of garlic derivatives administration. The intracellular localization of β-catenin was examined with immunofluorescent confocal microscopy. Results: A dual role of DADS, dependent on the duration of treatment, was revealed. Acute administration induced a significant decrease in invasive potential, while prolonged treatment promoted HER2+ cell invasiveness. These effects were directly correlated with the activation of Akt and the nuclear accumulation of β-catenin, known to induce expression of genes associated with tumor malignancy. Conclusions: Although further investigations are needed to establish the exact mechanism and to assess the in vivo reproducibility of these phenomena, our results highlight the heterogeneous response to natural compounds of complex diseases like cancer.

Gait disorders are the most frequent symptoms associated to multiple sclerosis (MS). Robot-assisted gait training (RAGT) in people with MS (PwMS) has been proposed as a possible effective treatment option for severe motor disability without significant superiority when compared to intensive overground gait training (OGT). Furthermore, RAGT at high intensity may enhance fatigue and spasticity. This study aims to evaluate the effects of a low-intensity RAGT at progressively increasing intensity compared to conventional RAGT and OGT in PwMS and moderate to severe walking impairment. 24 PwMS will be recruited and assigned to one of the three treatment groups: low-intensity RAGT at progressively increasing intensity, conventional RAGT and OGT. All participants will receive 3-weekly treatment sessions of 3 hours each for 4 weeks. In the first 2 hours of treatment, all participants will receive a rehabilitation programme based on stretching exercises, muscle strengthening and educational interventions. During the last hour, subjects will undergo specific gait training according to the assignment group. Outcomes will be assessed before and after treatment and at 3-month follow-up. The primary outcome is walking speed. Secondary outcomes include mobility and balance, psychological measures, muscle oxygen consumption, electrical and haemodynamic brain activity, urinary biomarkers, usability, and acceptability of robotic devices for motor rehabilitation. The results of this study will provide a safe, affordable and non-operator-dependent, intervention for PwMS. Results in terms of functional, psychological, neurophysiological and biological outcomes will confirm our hypothesis. The study’s trial registration number: NCT06381440.

Transglutaminase 2 (TG2) performs many functions both under physiological and pathological conditions. In cancer, its expression is associated with aggressiveness, propensity to epithelial-mesenchymal transition, and metastasis. Since TG2 performs key functions both outside and inside the cell, using inhibitors with different membrane permeability we analyzed the changes in the transcriptome induced in two triple-negative cell lines (MDA-MB-436 and MDA-MB-231) with aggressive features. By characterizing pathways and gene networks, we were able to define the effects of TG2 inhibitors (AA9, membrane-permeable, and NCEG2, impermeable) in relation to the roles of the enzyme in the intra- and extracellular space within the context of breast cancer.The deregulated genes revealed p53 and integrin signaling to be the common pathways with some genes showing opposite changes in expression. In MDA-MB-436, AA9 induced apoptosis, modulated cadherin, Wnt, gastrin and cholecystokinin receptors (CCKR) mediated signaling, with RHOB and GNG2 playing significant roles, and affected the Warburg effect by decreasing glycolytic enzymes. In MDA-MB-231 cells, AA9 strongly impacted HIF-mediated hypoxia, including AKT and mTOR pathway. These effects suggest an anti-tumor activity by blocking intracellular TG2 functions. Conversely, the use of NCEG2 stimulated the expression of ATP synthase and proteins involved in DNA replication, indicating a potential promotion of cell proliferation through inhibition of extracellular TG2. To effectively utilize these molecules as an anti-tumor strategy, an appropriate delivery system should be evaluated to target specific functions and avoid adverse effects. Additionally, considering combinations with other pathway modulators is crucial.

Breast cancer includes tumor subgroups with morphological, molecular, and clinical differences. Intrinsic heterogeneity especially characterizes breast tumors with a triple negative phenotype, often leading to the failure of even the most advanced therapeutic strategies. To improve breast cancer treatment, the use of natural agents to integrate conventional therapies is the subject of ever-increasing attention. In this context, garlic (Allium sativum) shows anti-cancerous potential, interfering with the proliferation, motility, and malignant progression of both non-invasive and invasive breast tumor cells. As heterogeneity could be at the basis of variable effects, the main objective of our study was to evaluate the anti-tumoral activity of a garlic extract in breast cancer cells with a triple negative phenotype. Established triple negative breast cancer (TNBC) cell lines from patient-derived xenografts (PDXs) were used, revealing subtype-dependent effects on morphology, cell cycle, and invasive potential, correlated with the peculiar down-modulation of Akt signaling, a crucial regulator in solid tumors. Our results first demonstrate that the effects of garlic on TNBC breast cancer are not unique and suggest that only more precise knowledge of the mechanisms activated by this natural compound in each tumor will allow for the inclusion of garlic in personalized therapeutic approaches to breast cancer.

Type 1 diabetes (T1D) is a chronic autoimmune condition characterized by the destruction of pancreatic β cells, recently estimated to affect approximately 8.75 million individuals worldwide. At variance with conventional management of T1D, which relies on exogenous insulin replacement and insulinotropic drugs, emerging therapeutic strategies include transplantation of insulin-producing cells (IPCs) derived from stem cells or fully reprogrammed differentiated cells. Through the in-depth analysis of the microRNAs (miRNAs) involved in the differentiation of human embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs), into insulin-producing cells, this review provides a comprehensive overview of the molecular mechanisms orchestrating the transformation of precursors to cells producing insulin. In addition to miR-375, involved in all differentiation processes, and to miR-7, mir-145 and miR-9, common to the generation of insulin-producing cells from at least two different sources, the literature reveals panels of miRNAs closely related to precursor cells and associated with specific events of the physiological β cell maturation. Since the forced modulation of miRNAs can direct cells development towards insulin-producing cells or modify their fate, a more comprehensive knowledge of the miRNAs involved in the cellular events leading to obtain efficient β cells could improve the diagnostic, prognostic, and therapeutic approaches to diabetes.

Convolvulus pluricaulis (CP) is a common Indian herb, largely employed in Ayurvedic medicine and known for its neuroprotective and neuroinflammatory action. Its effectiveness against several pathologic/sub-pathologic conditions is widely accepted, but it is not yet completely chemically characterized. In recent years, several researchers have pointed out the involvement of CP and other Convolvulaceae in lipidic and glucidic metabolism, particularly in the control of hyperlipidaemia and diabetic conditions. In this scenario, the aim of the study was to chemically characterize the medium polarity part of the CP whole plant and its fractions and to shed light on their biological activity in adipocyte differentiation using the 3T3-L1 cell model. Our results demonstrated that the CP extract and fractions could upregulate the adipocyte differentiation through the modulation of the nuclear receptor PPARγ (Peroxisome Proliferator-Activated Receptor γ), broadly recognized as a key regulator of adipocyte differentiation, and the glucose transporter GLUT-4, which is fundamental for cellular glucose uptake and for metabolism control. CP also showed the ability to exert an anti-inflammatory effect, downregulating cytokines such as Rantes, MCP-1, KC, eotaxin, and GM-CSF, which are deeply involved in insulin resistance and glucose intolerance. Taken together, these data suggest that CP could exert a potential beneficial effect on glycemia and could be employed as an anti-diabetic adjuvant or, in any case, a means to better control glucose homeostasis.

Voghiera garlic is an Italian white garlic variety which obtained in 2010 the Protected Designation of Origin. It is widely used for culinary purposes or as an ingredient for supplement production due to its phytochemical compositions. The storage conditions seem to be crucial to retain the high quality of garlic bulbs and their by-products, taking into account the high importance of organosulfur and phenolic compounds for the bioactive potency of garlic and its shelf-life. This study aims to examine the effect of storage on the phytochemical composition, biological effects, and shelf-life of Voghiera garlic PDO. In detail, we considered (i) −4 °C (industrial storage) for 3, 6, and 9 months; (ii) +4 °C for 3 months (home conservation), and (iii) −4 °C for 3 months, plus +4 °C for another 3 months. We focused our attention on the organosulfur compounds, total condensed tannins, flavonoids, phenolic compounds, and related antioxidant activity changes during the storage period. To evaluate the bioactive effects, the Voghiera garlic extracts at different storage conditions were administered to a breast cancer cell line, while antioxidant and anti-inflammatory activity was detected using macrophage RAW 264.7 cells. We observed a decrease in sulfur compounds after 6 months which correlated to a decrease in bioactive effects, while the number of antioxidant compounds was stable during the storage period, showing the good effect of refrigerated temperature in maintaining garlic bulb shelf-life.

The role of transglutaminase type 2 in cell physiology is related to protein transamidation and signal transduction (affecting extracellular, intracellular and nuclear processes) aided by the expression of truncated isoforms and of two lncRNAs with regulatory functions.In breast cancer TG2 is associated with disease progression supporting motility, epithelial-mesenchymal transition, invasion and drug resistance.The aim of his work is to clarify these issues by emphasizing the interconnections among TGM2 variants and transcription factors associated with an aggressive phenotype, in which the truncated TGH isoform correlates with malignancy.TGM2 transcripts are upregulated by several drugs in MCF-7, but only Doxorubicin is effective in MDA-MB-231 cells.These differences reflect the expression of GATA3, as demonstrated by silencing, suggesting a link between this transcription factor and gene dysregulation.Of note, NC9, an irreversible inhibitor of enzymatic TG2 activities, emerges to control NF-ĸB and apoptosis in breast cancer cell lines, showing potential for combination therapies with Doxorubicin.

Since the multifunctionality of transglutaminase 2 (TG2) includes extra- and intracellular functions, we investigated the effects of intracellular administration of TG2 inhibitors in three breast cancer cell lines, MDA-MB-231, MDA-MB-436 and MDA-MB-468, which are representative of different triple-negative phenotypes, using a patch-clamp technique. The first cell line has a highly voltage-dependent a membrane current, which is low in the second and almost absent in the third one. While applying a voltage protocol to responsive single cells, injection of TG2 inhibitors triggered a significant decrease of the current in MDA-MB-231 that we attributed to voltage-dependent K+ channels using the specific inhibitors 4-aminopyridine and astemizole. Since the Kv10.1 channel plays a dominant role as a marker of cell migration and survival in breast cancer, we investigated its relationship with TG2 by immunoprecipitation. Our data reveal their physical interaction affects membrane currents in MDA-MB-231 but not in the less sensitive MDA-MB-436 cells. We further correlated the efficacy of TG2 inhibition with metabolic changes in the supernatants of treated cells, resulting in increased concentration of methyl- and dimethylamines, representing possible response markers. In conclusion, our findings highlight the interference of TG2 inhibitors with the Kv10.1 channel as a potential therapeutic tool depending on the specific features of cancer cells.

The miR-29 family comprises miR-29a, miR-29b, and miR-29c, and these molecules play crucial and partially overlapped functions in solid tumors, in which the different isoforms are variously de-regulated and mainly correlated with tumor suppression. miR-29b is the most expressed family member in cancer, in which it is involved in regulating gene expression at both transcriptional and post-transcriptional levels. This review focuses on the role of miR-29b in breast cancer, in which it plays a controversial role as tumor suppressor or onco-miRNA. Here we have highlighted the dual effect of miR-29b on breast tumor features, which depend on the prevailing function of this miRNA, on the mature miR-29b evaluated, and on the breast tumor characteristics. Remarkably, the analyzed miR-29b form emerged as a crucial element in the results obtained by various research groups, as the most abundant miR-29b-3p and the less expressed miR-29b1-5p seem to play distinct roles in breast tumors with different phenotypes. Of particular interest are the data showing that miR-29b1-5p counteracts cell proliferation and migration and reduces stemness in breast tumor cells with a triple negative phenotype. Even if further studies are required to define exactly the role of each miR-29b, our review highlights its possible implication in phenotype-specific management of breast tumors.

Triple negative breast cancer (TNBC) represents the most aggressive breast tumor, showing a high intrinsic variability in terms of both histopathological features and response to therapies. Blocking the Akt signaling pathway is a well-studied approach in the treatment of aggressive breast tumors. The high homology among the Akt isoforms and their distinct, and possibly opposite, oncogenic functions made it difficult to develop effective drugs. Here we investigated the role of Vav1 as a potential down-regulator of individual Akt isozymes. We revealed that the over-expression of Vav1 in triple negative MDA-MB-231 cells reduced only the Akt2 isoform, acting at the post-transcriptional level through the up-modulation of miR-29b. The Vav1/miR-29b dependent decrease in Akt2 was correlated with a reduced lung colonization of circulating MDA-MB-231 cells. In cell lines established from PDX, the Vav1 induced down-modulation of Akt2 is strongly dependent on miR-29b and occurs only in some TNBC tumors. These findings may contribute to better classify breast tumors having the triple negative phenotype, and suggest that the activation of the Vav1/miR-29b axis, precisely regulating the amount of an Akt isozyme crucial for tumor dissemination, could have great potential for driving more accurate therapies to TNBCs, often not eligible or resistant to treatments.

Background: In breast cancer, low oxygen availability is associated with a more aggressive phenotype and with malignant evolution of non-invasive cells. Natural compounds have long attracted attention in cancer treatment, and in recent years garlic (Allium sativum) organosulfur derivatives have been shown to negatively affect growth and invasion of tumor cells. Methods: Homemade ethanol-based garlic extract (GE) was administered to MCF7 and MCF10DCIS breast tumor cell lines grown under moderate hypoxia. Cell cycle, epithelial-to-mesenchymal transition and cancer stem cell markers were evaluated. Results: We revealed that, in the non-invasive MCF10DCIS cells but not in the post-EMT MCF7 cells, low oxygen availability induced the decrease of E-cadherin and the increase of vimentin and motility, that were prevented by GE administration. In both cell lines, treatment with GE counteracted the up-modulation of CD133 positive cells induced by hypoxia. Conclusions: Overall, our data firstly revealed anti-cancer properties of garlic in non-invasive breast cancer cells. In particular, they demonstrated a protective role of this natural product against the hypoxia-induced increase of molecules that play crucial roles in tumor evolution, suggesting that garlic derivatives can be considered in new approaches for preventing progression of breast tumors from non-invasive to infiltrating lesions.

Drug resistance is one of the major forces driving a poor prognosis during the treatment and progression of human colon carcinomas. The molecular mechanisms that regulate the diverse processes underlying drug resistance are still under debate. MicroRNAs (miRNAs) are a subgroup of non-coding RNAs increasingly found to be associated with the regulation of tumorigenesis and drug resistance. We performed a systematic review of the articles concerning miRNAs and drug resistance in human colon cancer published from 2013 onwards in journals with an impact factor of 5 or higher. First, we built a network with the most studied miRNAs and targets (as nodes) while the drug resistance/s are indicated by the connections (edges); then, we discussed the most relevant miRNA/targets interactions regulated by drugs according to the network topology and statistics. Finally, we considered the drugs as nodes in the network, to allow an alternative point of view that could flow through the treatment options and the associated molecular pathways. A small number of microRNAs and proteins appeared as critically involved in the most common drugs used for the treatment of patients with colon cancer. In particular, the family of miR-200, miR34a, miR-155 and miR-17 appear as the most relevant microRNAs. Thus, regulating these miRNAs could be useful for interfering with some drug resistance mechanisms in colorectal carcinoma.