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Cigarette smoke is associated to severe chronic diseases. The most harmful components of cigarette smoke derive from the combustion process, which are significantly reduced in the electronic cigarette aerosol, thus providing a valid option in harm reduction strategies. To develop safer products, it is therefore necessary to screen electronic cigarette liquids (e-liquids) to meet high safety standards defined by government regulations. The aim of the present study was to evaluate the presence of metal- and plastic-derived contaminants in four different commercial e-liquids with high concentration of nicotine and their cytotoxic effect in normal human bronchial epithelial cells by a number of in vitro assays, in comparison with the 1R6F reference cigarette, using an air-liquid interface (ALI) exposure system. Moreover, we evaluated the effect of aerosol exposure on oxidative stress by measuring the production of reactive oxygen species and mitochondrial potential. Our results showed no contaminants in all e-liquids and a significantly reduced cytotoxic effect of e-liquid aerosol compared to cigarette smoke as well as a maintained mitochondria integrity. Moreover, no production of reactive oxygen species was detected with e-cigarette aerosol. In conclusion, these results support the reduced toxicity potential of e-cigs compared to tobacco cigarettes in an in vitro model resembling real life smoke exposure.

Our earlier studies had shown that overexpression of the JmjC domain histone demethylase KDM2B renders mouse embryo fibroblasts (MEFs) resistant to oxidative stress due to the role of KDM2B in the regulation of antioxidant mechanisms. Here we present evidence that the knockdown of KDM2B in basal-like breast cancer cell lines results in a decrease of Glutathione (GSH) levels, a secondary increase of intracellular ROS levels and in enhanced sensitivity to deubiquitinase inhibitors. The expression of the Glutamate-Cystine antiporter, the Glutamate-Cysteine Ligase GCLC/GCLM and the Glutathione Peroxidase GPX4, all of which regulate GSH abundance was not affected. To address the mechanism of the GSH regulation we carried out RNA-Seq, quantitative proteomics and metabolomics analyses in shKDM2B- and empty vector-transduced MDA-MB-231 cells. The results showed that the KD of KDM2B causes major shifts in metabolism and that one of the metabolic pathways whose activity depends on KDM2B is the SGOC pathway, which has a major role in GSH biosynthesis. Experiments in cultured cells confirmed the importance of KDM2B in the regulation of this pathway and they also showed that the inhibition of the pathway via the KD of KDM2B is partly responsible for the shKDM2B-induced inhibition of cell proliferation in culture and in xenograft experiments in NSG mice. More important, the transcriptomic signature of the SGOC pathway correlates with the expression of KDM2B in basal like mammary adenocarcinomas in the TCGA database. The genes encoding the majority of the enzymes in the SGOC pathway are known to be regulated by MYC and ATF4 and our data show that both MYC and ATF4 are under the regulatory control of KDM2B. ATAC-Seq and ChIP-Seq experiments in shKDM2B and control MDA-MB-231 cells also showed that KDM2B binds the promoter region of not only MYC and ATF4, but also of the genes encoding the SGOC enzymes and that it regulates chromatin accessibility and the abundance of H3K4me3/H3K27Ac active histone marks in these promoters. Overall, our data indicate that KDM2B regulates the SGOC pathway by targeting MYC and ATF4 and by making the promoters of the genes encoding the SGOC enzymes accessible to these regulators. Overall, our data provide new evidence on SGOC regulation, and identify novel KDM2B-dependent metabolic vulnerabilities in basal like breast cancer. Citation Format: Evangelia Chavdoula, Vollter Anastas, Allesandro La Ferlita, Julian Aldana, Giuseppe Carota, Mariarita Spampinato, Sameer Parashar, Ilaria Cosentini, Burak Soysal, Giovanni Nigita, Michael Freitas, Philip Tsichlis. KDM2B regulates Serine-Glycine-One Carbon (SGOC) metabolism by targeting the SGOC enzyme genes via a combination of direct and indirect epigenetic mechanisms. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6038.

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Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related death mainly due to its high metastatic rate. Impairment of redox homeostasis mechanisms has been previously described in NSCLC and is associated with the disease itself as well as with comorbidities such as smoking. The aim of the present in vitro study was to evaluate the effect of selective and non-competitive inhibition of heme oxygenase-1 (HO-1) on cancer redox homeostasis with particular regards to glutathione (GSH) metabolism related enzymes. NSCLC cell line (A549) was treated with the HO-1 activity inhibitor VP13/47 (10 µM) and we further evaluated cell viability, apoptosis, mitochondrial dysfunction and oxidative stress. Our results showed that VP13/47 significantly reduced HO-1 expression and total HO activity thus, resulting in a significant reduction of cell viability, proliferation and increased apoptosis, mitochondrial dysfunction and oxidative stress. Consistently with increased oxidative stress, we also showed that reduced GSH was significantly decreased and such effect was also accompanied by a significant downregulation of the enzymes involved in its biosynthesis. Taken all together our results show that selective HO-1 inhibition significantly impairs NSCLC progression and may represent a possible pharmacological strategy for new chemotherapy agents.

Although the epidemic caused by SARS-CoV-2 callings for international attention to develop new effective therapeutics, no specific protocol is yet available, leaving patients to rely on general and supportive therapies. A range of respiratory diseases, including pulmonary fibrosis, have been associated with higher iron levels that may promote the course of viral infection. Recent studies have demonstrated that some natural components could act as the first barrier against viral injury by affecting iron metabolism. Moreover, a few recent studies have proposed the combination of protease inhibitors for therapeutic use against SARS-CoV-2 infection, highlighting the role of viral protease in virus infectivity. In this regard, this review focuses on the analysis, through literature and docking studies, of a number of natural products able to counteract SARS-CoV-2 infection, acting both as iron chelators and protease inhibitors.

Electronic Nicotine Delivery Systems (ENDS), i.e., electronic cigarettes (e-cigs) and Tobacco Heating Products (THPs), are rapidly growing in popularity. The marketing of these products is regulated by specific rules in the European Union and in the US, which permit their legal sales. Nonetheless, comprehensive quality and safety requirements for regulatory purposes are still under development. Cytotoxicity studies are an important initial step in appraising the potential toxicity of ENDS. The aim of the present study was to screen a battery of different in vitro cytotoxicity methods for the assessment of toxicity induced by ENDS. We evaluated different cytotoxicity assays, including neutral red uptake (NRU), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Annexin V apoptosis, High Content Screening (HCS) assays and Real Time Cell Analysis (RTCA), to compare two e-cigs (Vype ePen 3 and Vype eStick Maxx) and two THPs (IQOS and GLO™) with the 1R6F reference tobacco cigarette. Human bronchial epithelial cells (H292) were exposed to 1R6F smoke (5 puffs by HCI regime), ePen vapor (10 puffs by modified HCI regime), eStick vapor (25 puffs by CRM81 regime), IQOS vapor (7 puffs by HCI regime) and GLO vapor (8 puffs by HCI regime) at air-liquid interface. All tests showed reduced cell viability following 1R6F smoke exposure and slight or no reduction with ENDS at 24 hours compared to controls. In addition, Annexin V and RTCA exhibited a further significant reduction in cell viability following 1R6F exposure compared with other assays. Furthermore, Annexin V allowed to discriminate viable cells from those in early/late apoptosis. Finally, RTCA and HCS being time-resolved analyses allowed also to determine the kinetic dependency parameter for toxicity of smoke/vapor chemicals on cell viability. In conclusion, NRU assay may be considered a suitable test, especially when combined with a time-resolved test, for assessing the kinetic of cytotoxicity induced by these products.

Lactobacillus strains dominate the vaginal habitat and they are associated with a lower risk of genital infections. In addition, they contribute to the conservation of the vaginal microbiota by producing postbiotic agents. Previous studies have shown that their predominance involves antimicrobial activity against urogenital pathologies. In this context, probiotics may improve treatment outcomes. The aim of this study was to evaluate the probiotic properties of lactobacilli strains of vaginal origin using a multidisciplinary approach. For this purpose, safety criteria, ability to resist at low pH and bile salts, antimicrobial activity, ability to produce biofilm, capacity to produce hydrogen peroxide and more importantly, auto-aggregation, co-aggregation (with Candida spp.) and adhesion to human cells were evaluated. The strains belonged to the species of L. crispatus, L. gasseri, L. rhamnosus and L. delbruckii. Among these, a strain of L. rhamnosus named AD3 showed the best probiotic properties. As probiotics are already in use in many clinical practice and there are no major safety concerns, L. rhamnosus AD3 showed promise in becoming a prevention and complementary treatment option for urogenital diseases. Indeed, these results suggest that strain L. rhamnosus AD3 is non-pathogenic and likely to be safe for human consumption. This study revealed the great amensalistic properties of a new L. rhamnosus strain which can aim to be used as probiotic in pharmaceutical applications.

The transition from prenatal to postnatal life causes a significant increase in arterial oxygen tension and the activation of metabolic pathways enabling the newborn's adaptation to the extra-uterine environment. The balance between pro-oxidant and anti-oxidant systems is critical to preserve cellular functions. Indeed, oxidative stress (OS) occurs when the production of free radicals is not balanced by the activity of intracellular antioxidant systems, contributing to cellular and tissue damage. Perinatal OS may have serious health consequences during the postnatal period and later in life. Namely, OS has been recognized as the major cause of lung injury in newborns, especially those preterm born, due to their immature lung and antioxidant systems. The development of OS biomarkers has gained increasing research interest since they may provide useful insights about pathophysiological pathways underlying OS-mediated pulmonary diseases in newborns. Moreover, their implementation in clinical settings may help to early identify high risk-newborns and to provide targeted treatment. Ideally, a biomarker should demonstrate ease of use, biological validity and reproducibility, high sensitivity and specificity. However, none of the clinically validated biomarkers so far have been qualified for neonatal lung disease. Additionally, the complex technical procedures and the high cost of such determinations have hampered the use of OS biomarkers in clinical practice. This review aims to evaluate the current evidence on the application of biomarkers of oxidative stress for neonatal lung disease and exploring the most relevant issues affecting their implementation in practice, as well as the associated evidence gaps and research limitations.

Recent experimental data suggest that fatty acids and lipotoxicity could play a role in the initiation and evolution of metabolic bone diseases such as osteoporosis. A functional bone marrow adipose tissue (BMAT) may provide support to surrounding cells and tissues or may serve as a lipid reservoir that protects skeletal osteoblasts from lipotoxicity. The present study examined the effect of N-acetylcysteine (NAC), a powerful antioxidant and precursor of glutathione, commonly used to treat chronic obstructive pulmonary disease, on triglycerides accumulation in bone marrow stromal cells-derived adipocytes. Quantification of Oil Red O stained cells showed that lipid droplets decreased following NAC treatment. Additionally, exposure of bone marrow stromal cells (HS-5) to NAC increased adiponectin, PPARγ, HO-1, and SIRT-1 and increased beta-oxidation markers such as PPARα and PPARδ mRNA levels. As there is now substantial interest in alternative medicine, the observed therapeutic value of NAC should be taken into consideration in diabetic patients.

Electronic Nicotine Delivery Systems (ENDS), i.e., electronic-cigarettes (e-cigs) and Tobacco Heating Products (THPs), are rapidly growing in popularity. Nonetheless, comprehensive quality and safety requirements for regulatory purposes are still under development. Cytotoxicity studies are important initial steps in appraising the potential ENDS toxicity. The aim of the present study was to screen different in vitro cytotoxicity methods for the assessment of ENDS toxicity. We evaluated NRU, MTT, Annexin V apoptosis (AN-V), High-Content Screening (HCS) assays and Real-Time Cell Analysis (RTCA), to compare two e-cigs and two THPs with the 1R6F reference tobacco cigarette. Human adenocarcinoma lung epithelial cells (H292) were exposed to tobacco smoke and ENDS vapor at air-liquid interface. All tests showed reduced cell viability following 1R6F smoke exposure and slight or no reduction with ENDS at 24 h. AN-V and RTCA exhibited a further significant reduction in cell viability following 1R6F exposure. AN-V allowed to discriminate viable cells from those in early/late apoptosis. RTCA and HCS being time-resolved analyses elucidate the kinetic dependency parameter for toxicity of smoke/vapor chemicals on cell viability. In conclusion, NRU assay may be considered a suitable test, especially when combined with a time-resolved analysis, for assessing the kinetic of cytotoxicity induced by these products.

Prevalence studies of current smoking among hospitalized COVID-19 patients demonstrated an unexpectedly low prevalence of current smoking among patients with COVID-19. The aim of the present proposal was to evaluate the effect of smoke from cigarettes on ACE-2 in bronchial epithelial cells. Normal bronchial epithelial cells (H292) were exposed to smoke by an air-liquid-interface (ALI) system and ACE-2 membrane protein expression was evaluated after 24 hours from exposure. Our transcriptomics data analysis showed a significant selective reduction of membrane ACE-2 expression (about 25%) following smoking exposure. Interestingly, we observed a positive direct correlation between ACE-2 reduction and nicotine delivery. Furthermore, by stratifying GSE52237 as a function of ACE-2 gene expression levels, we highlighted 1012 genes related to ACE-2 in smokers and 855 in non-smokers. Furthermore, we showed that 161 genes involved in the endocytosis process were highlighted using the online pathway tool KEGG. Finally, 11 genes were in common between the ACE-2 pathway in smokers and the genes regulated during endocytosis, while 12 genes with non-smokers. Interestingly, six in non-smokers and four genes in smokers were closely involved during the viral internalization process. Our data may offer a pharmaceutical role of nicotine as potential treatment option in COVID-19.

Lemon fruit is a source of bioactive compounds, which has many beneficial effects on health. Obesity is characterized by over-accumulation of adipose tissue as a result of increased adipocyte size and number. Adipogenesis is mediated and assisted by various transcription factors that induce lipid-metabolizing enzymes followed by an increase of perilipin expression and lipid droplets generation. Here, we evaluate the effect of lemon extract (LE) as radical scavenger and the consequent regulation of adipocyte differentiation and lipid accumulation. 3T3-L1 murine pre-adipocytes were differentiated and treated with different LE concentrations. The high percentages of flavonoid contained in LE led to a significant inhibition of DPPH radical and reactive oxygen species, demonstrating a strong radical scavenger activity. Treatment of 3T3-cells with LE showed a significant decrease of perilipin expression, subsequently confirmed by the reduction of lipid droplet accumulation, resulting from Oil Red O Staining and by the downregulation of PPARγ and DGAT-1mRNA.

In recent years, there has been a growth in scientific interest in nutraceuticals, which are those nutrients in foods that have beneficial effects on health. Nutraceuticals can be extracted, used for food supplements, or added to foods. There has long been interest in the antiviral properties of nutraceuticals, which are especially topical in the context of the ongoing COVID-19 pandemic. Therefore, the purpose of this review is to evaluate the main nutraceuticals to which antiviral roles have been attributed (either by direct action on viruses or by modulating the immune system), with a focus on the pediatric population. Furthermore, the possible applications of these substances against SARS-CoV-2 will be considered.

Background:Prostate Cancer (PC) represents a leading cause of tumor-related death among men in the Western world. Above all, DU145 cell line represents the most particular cells model of PC, derived from a central nervous system metastasis. In recent years, functional and healthy diet has gained a pivotal role in society, allowing the possibility to deal with cancer before its emergence or progression, profiting by anti-tumor properties of dietary phytochemicals. Among them, Ellagic Acid (EA) is found in several fruits and vegetables, whose juice demonstrated antioxidant, anti-carcinogenic and anti-fibrotic properties.Methods:DU145 prostate cancer cell line was used to determine the effects of ellagic acid on cell viability. In order to evaluate metastatic feature of DU145, VEGF-A and OPG levels by ELISA assay were assessed. Expression of β-catenin, HO-1, HO-2 and SIRT1, markers of proliferative and defense capacities, were determined by western blotting. To strengthen the study, cell transfection with siRNA β-catenin was performed.Results:In the presence of EA, the viability of DU145 cells was reduced by about 40 and 50%, respectively after the exposure to 50 and 100 μM concentrations. We also observed a reduction of both levels of VEGF-A and OPG, confirming the important role of EA in facing the metastasis development. EA treatment (50 μM) induced a significant reduction of β-catenin and SIRT1 levels and, similarly, there was a decrease of HO protein expression, more pronounced for HO-2, showing EA activity on the proliferative feature of DU145 cells. Knockdown of β-catenin by siRNA, in the presence of EA treatment, inhibited cell proliferation.Conclusion:Ellagic acid exhibits significant antiproliferative effects in ourin vitromodel of prostate cancer’s metastasis, suggesting that, the use of EA as a multitarget natural compound, may represent a possible strategy for cancer chemoprevention.

High levels of heme oxygenase (HO)-1 have been frequently reported in different human cancers, playing a major role in drug resistance and regulation of cancer cell redox homeostasis. Metformin (MET), a drug widely used for type 2 diabetes, has recently gained interest for treating several cancers. Recent studies indicated that the anti-proliferative effects of metformin in cancer cells are highly dependent on glucose concentration. The present work was directed to determine whether use of a specific inhibitor of HO-1 activity, alone or in combination with metformin, affected metastatic prostate cancer cell viability under different concentrations of glucose. MTT assay and the xCELLigence system were used to evaluate cell viability and cell proliferation in DU145 human prostate cancer cells. Cell apoptosis and reactive oxygen species were analyzed by flow cytometry. The activity of HO-1 was inhibited using a selective imidazole-based inhibitor; genes associated with antioxidant systems and cell death were evaluated by qRT-PCR. Our study demonstrates that metformin suppressed prostate cancer growth in vitro and increased oxidative stress. Disrupting the antioxidant HO-1 activity, especially under low glucose concentration, could be an attractive approach to potentiate metformin antineoplastic effects and could provide a biochemical basis for developing HO-1-targeting drugs against solid tumors.

Heme oxygenase (HO) enzymes are involved in heme catabolism and several physiological functions. Among the different HO isoforms, HO-2 stands out for its neuroprotective properties and modulatory activity in male reproduction. However, unlike the HO-1 ligands, the potential therapeutic applications of HO-2 inhibitors/activators have not been extensively explored yet. Moreover, the physiological role of HO-2 is still unclear, mostly due to the lack of highly selective HO-2 chemical probes. To boost the interest on this intriguing target, the present review updates the knowledge on the structure-activity relationships of HO-2 inhibitors and activators, as well as their potential therapeutic applications. To the best of our knowledge, among HO-2 inhibitors, clemizole derivatives are the most selective HO-2 inhibitors reported so far (IC50 HO-1 >100 μM, IC50 HO-2 = 3.4 μM), while the HO-2 nonselective inhibitors described herein possess IC50 HO-2 values ≤ 10 μM. Furthermore, the development of HO-2 activators, such as menadione analogues, helped to understand the critical moieties required for HO-2 activation. Recent advances in the potential therapeutic applications of HO-2 inhibitors/activators cover the fields of neurodegenerative, cardiovascular, inflammatory, and reproductive diseases further stimulating the interest towards this target.

Ginseng is an ancient herb, belonging to Asian traditional medicine, that has been considered as a restorative to enhance vitality for centuries. It has been demonstrated that the antioxidant action of ginseng may be mediated through activation of different cellular signaling pathways involving the heme oxygenase (HO) system. Several compounds derived from ginseng have been studied for their potential role in brain, heart and liver protection, and the Nrf2 pathway seems to be the most affected by these natural molecules to exert this effect. Ginseng is also popularly used in cancer patients therapy for the demonstrated capability to defend tissues from chemotherapy-induced damage. Reported results suggest that the effect of ginseng is primarily associated with ROS scavenging, mainly exerted through the activation of Nrf2 pathway, and the consequent induction of HO-1 levels. This review aims to discuss the connection between the antioxidant properties of ginseng and the activation of the HO system, as well as to outline novel therapeutic applications of this medicinal plant to human health.