Novel Acetamide-Based HO-1 Inhibitor Counteracts Glioblastoma Progression by Interfering with the Hypoxic-Angiogenic Pathway.

Glioblastoma multiforme (GBM) represents the deadliest tumor among brain cancers. It is a solid tumor characterized by uncontrolled cell proliferation generating the hypoxic niches in the cancer core. By inducing the transcription of hypoxic inducible factor (HIF), hypoxia triggers many signaling cascades responsible for cancer progression and aggressiveness, including enhanced expression of vascular endothelial growth factor (VEGF) or antioxidant enzymes, such as heme oxygenase-1 (HO-1). The present work aimed to investigate the link between HO-1 expression and the hypoxic microenvironment of GBM by culturing two human glioblastoma cell lines (U87MG and A172) in the presence of a hypoxic mimetic agent, deferoxamine (DFX). By targeting hypoxia-induced HO-1, we have tested the effect of a novel acetamide-based HO-1 inhibitor (VP18/58) on GBM progression. Results have demonstrated that hypoxic conditions induced upregulation and nuclear expression of HO-1 in a cell-dependent manner related to malignant phenotype. Moreover, our data demonstrated that the HO-1 inhibitor counteracted GBM progression by modulating the HIFα/HO-1/VEGF signaling cascade in cancer cells bearing more malignant phenotypes.

Correction: Innovative snail-mucus-extract (SME)-coated nanoparticles exhibit anti-inflammatory and anti-proliferative effects for potential skin cancer prevention and treatment.

[This corrects the article DOI: 10.1039/D4RA00291A.].

Synthesis and in Vitro Evaluation of CAPE Derivatives as Ferroptosis Inducers in Triple Negative Breast Cancer.

Herein, we describe the design, synthesis, and in vitro biological evaluation of HO-1 inducers endowed with cytotoxic effects mediated by ferroptosis activation. Using the natural HO-1 inducer caffeic acid phenethyl ester (CAPE) as a chemical scaffold, new derivatives were synthesized by performing modifications in the cathecol moiety and in the phenethyl ester aromatic ring. Biological assays aimed at evaluating an imbalanced activity of ferroptosis key players identified that 2-(1H-indol-3-yl)ethyl cinnamate (compound 24) possesses improved anticancer activity toward the MDA-MB 231 triple negative breast cancer cell line when compared to CAPE. Increased ROS and LOOH levels, reduced GSH levels, imbalanced mitochondrial activity, and restored cell viability after ferrostatin-1 treatment suggested a ferroptotic mechanism of action, which did not involve GPX4 inhibition. Compound 24 represents an intriguing hit compound useful for the identification of novel ferroptosis inducers.

Evaluation of the anticancer effects exerted by 5-fluorouracil and heme oxygenase-1 inhibitor hybrids in HTC116 colorectal cancer cells.

Colon cancer remains a clinical challenge in industrialised countries. Its treatment with 5-Flurouracil (5-FU) develops many side effects and resistance. Thus, several strategies have been undertaken so far, including the use of drug cocktails and polypharmacology. Heme oxygenase-1 (HO-1) is an emerging molecular target in the treatment of various cancers. We recently demonstrated that a combination of HO-1 inhibitors with 5-FU and the corresponding hybrids SI1/17, SI1/20, and SI1/22, possessed anticancer activity against prostate and lung cancer cells. In this work, we evaluated these hybrids in a model of colon cancer and found that SI1/22 and the respective combo have greater potency than 5-FU. Particularly, compounds inhibit HO-1 activity in cell lysates, increase ROS and the expression of HO-1, SOD, and Nrf2. Moreover, we observed a decrease of pro-caspase and an increase in cleaved PARP-1 and p62, suggesting apoptotic and autophagic cell death and potential application of these drugs as anticancer agents.

Improvement of hepatic innate immunity in chemically-injured livers to develop hepatocarcinoma by a serine type-protease inhibitors enriched extract from Chenopodium quinoa.

Food ingredients have critical effects on the maturation and development of the immune system, which innate - lymphoid (ILCs) and myeloid - cells play key roles as important regulators of energy storage and hepatic fat accumulation. Therefore, the objective of this study is to define potential links between a dietary immunonutritional induction of the selective functional differentiation of monocytes-derived macrophages, ILCs and lipid homeostasis in hepatocarcinoma (HCC)-developing mice. Hepatic chemically injured (diethylnitrosamine/thiacetamide) Rag2-/- and Rag2-/-Il2-/- mice were administered with serine-type protease inhibitors (SETIs) obtained from Chenopodium quinoa. Early HCC-driven immunometabolic imbalances (infiltrated macrophages, glucose homeostasis, hepatic lipid profile, ILCs expansion, inflammatory conditions, microbiota) in animals put under a high-fat diet for 2 weeks were assessed. It was also approached the potential of SETIs to cause functional adaptations of the bioenergetics of human macrophage-like cells (hMLCs) in vitro conditioning their capacity to accumulate fat. It is showed that Rag2-/-Il2-/- mice, lacking ILCs, are resistant to the SETIs-induced hepatic macrophages (CD68+F4/80+) activation. Feeding SETIs to Rag2-/- mice, carrying ILCs, promoted the expansion towards ILC3s (CD117+Nkp46+CD56+) and reduced that of ILC2s (CD117+KLRG1+) into livers. In vitro studies demonstrate that hMLCs, challenged to SETIs, develop a similar phenotype of that found in mice and bioenergetic adaptations leading to increased lipolysis. It is concluded that SETIs promote liver macrophage activation and ILCs adaptations to ameliorate HCC-driven immunometabolic imbalances.

Novel Insights on Ferroptosis Modulation as Potential Strategy for Cancer Treatment: When Nature Kills.

Significance: The multifactorial nature of the mechanisms implicated in cancer development still represents a major issue for the success of established antitumor therapies. The discovery of ferroptosis, a novel form of programmed cell death distinct from apoptosis, along with the identification of the molecular pathways activated during its execution, has led to the uncovering of novel molecules characterized by ferroptosis-inducing properties. Recent advances: As of today, the ferroptosis-inducing properties of compounds derived from natural sources have been investigated and interesting findings have been reported both in vitro and in vivo. Critical Issues: Despite the efforts made so far, only a limited number of synthetic compounds have been identified as ferroptosis inducers, and their utilization is still limited to basic research. In this review, we analyzed the most important biochemical pathways involved in ferroptosis execution, with particular attention to the newest literature findings on canonical and non-canonical hallmarks, together with mechanisms of action of natural compounds identified as novel ferroptosis inducers. Compounds have been classified based on their chemical structure, and modulation of ferroptosis-related biochemical pathways has been reported. Future Directions: The outcomes herein collected represent a fascinating starting point from which to take hints for future drug discovery studies aimed at identifying ferroptosis-inducing natural compounds for anticancer therapies. Antioxid. Redox Signal. 40, 40-85.

Evaluation of the Antioxidant and Antiangiogenic Activity of a Pomegranate Extract in BPH-1 Prostate Epithelial Cells.

Benign prostatic hypertrophy (BPH) is a noncancerous enlargement of the prostate gland that develops from hyper-proliferation of the stromal and epithelium region. Activation of pathways involving inflammation and oxidative stress can contribute to cell proliferation in BPH and tumorigenesis. Agricultural-waste-derived extracts have drawn the attention of researchers as they represent a valid and sustainable way to exploit waste production. Indeed, such extracts are rich in bioactive compounds and can provide health-promoting effects. In particular, extracts obtained from pomegranate wastes and by-products have been shown to exert antioxidant and anti-inflammatory effects. This study focused on the evaluation of the anti-angiogenic effects and chemopreventive action of a pomegranate extract (PWE) in cellular models of BPH. In our experimental conditions, we observed that PWE was able to significantly (p < 0.001) reduce the proliferation and migration rates (up to 60%), together with the clonogenic capacity of BPH-1 cells concomitantly with the reduction in inflammatory cytokines (e.g., IL-6, PGE2) and pro-angiogenic factor (VEGF-ADMA) release. Additionally, we demonstrated the ability of PWE in reducing angiogenesis in an in vitro model of BPH consisting in transferring BPH-1-cell-conditioned media to human endothelial H5V cells. Indeed, PWE was able to reduce tube formation in H5V cells through VEGF level reduction even at low concentrations. Overall, we confirmed that inhibition of angiogenesis may be an alternative therapeutic option to prevent neovascularization in prostate tissue with BPH and its transformation into malignant prostate cancer.

Standardized Extract from Wastes of Edible Flowers and Snail Mucus Ameliorate Ultraviolet B-Induced Damage in Keratinocytes.

Several studies have highlighted the ability of snail mucus in maintaining healthy skin conditions due to its emollient, regenerative, and protective properties. In particular, mucus derived from Helix aspersa muller has already been reported to have beneficial properties such as antimicrobial activity and wound repair capacity. In order to enhance the beneficial effects of snail mucus, a formulation enriched with antioxidant compounds derived from edible flower waste (Acmella oleracea L., Centaurea cyanus L., Tagetes erecta L., Calendula officinalis L., and Moringa oleifera Lam.) was obtained. UVB damage was used as a model to investigate in vitro the cytoprotective effects of snail mucus and edible flower extract. Results demonstrated that polyphenols from the flower waste extract boosted the antioxidant activity of snail mucus, providing cytoprotective effects in keratinocytes exposed to UVB radiation. Additionally, glutathione content, reactive oxygen species (ROS), and lipid peroxidation levels were reduced following the combined treatment with snail mucus and edible flower waste extract. We demonstrated that flower waste can be considered a valid candidate for cosmeceutical applications due to its potent antioxidant activity. Thus, a new formulation of snail mucus enriched in extracts of edible flower waste could be useful to design innovative and sustainable broadband natural UV-screen cosmeceutical products.

Discovery of SI 1/20 and SI 1/22 as Mutual Prodrugs of 5-Fluorouracil and Imidazole-Based Heme Oxygenase 1 Inhibitor with Improved Cytotoxicity in DU145 Prostate Cancer Cells.

In this work, we extend the concept of 5-fluorouracil/heme oxygenase 1 (5-FU/HO-1) inhibitor hybrid as an effective strategy for enhancing 5-FU-based anticancer therapies. For this purpose, we designed and synthesized new mutual prodrugs, named SI 1/20 and SI 1/22, in which the two active parent drugs (i. e., 5-FU and an imidazole-based HO-1 inhibitor) were connected through an easily cleavable succinic linker. Experimental hydrolysis rate, and in silico ADMET predictions were indicative of good drug-likeness and pharmacokinetic properties. Novel hybrids significantly reduced the viability of prostate DU145 cancer cells compared to the parent compounds 5-FU and HO-1 inhibitor administered alone or in combination. Interestingly, both compounds showed statistically significant lower toxicity, than 5-FU at the same dose, against non-tumorigenic human benign prostatic hyperplasia (BPH-1) cell line. Moreover, the newly synthesized mutual prodrugs inhibited the HO-1 activity both in a cell-free model and in vitro, as well as downregulated the HO-1 expression and increased the reactive oxygen species (ROS) levels.

Recent Advances in Health Benefits of Bioactive Compounds from Food Wastes and By-Products: Biochemical Aspects.

Bioactive compounds, including terpenoids, polyphenols, alkaloids and other nitrogen-containing constituents, exert various beneficial effects arising from their antioxidant and anti-inflammatory properties. These compounds can be found in vegetables, fruits, grains, spices and their derived foods and beverages such as tea, olive oil, fruit juices, wine, chocolate and beer. Agricultural production and the food supply chain are major sources of food wastes, which can become resources, as they are rich in bioactive compounds. The aim of this review is to highlight recent articles demonstrating the numerous potential uses of products and by-products of the agro-food supply chain, which can have various applications.

Optimization of Lipid Nanoparticles by Response Surface Methodology to Improve the Ocular Delivery of Diosmin: Characterization and In-Vitro Anti-Inflammatory Assessment.

Diosmin is a flavonoid with a great variety of biological activities including antioxidant and anti-inflammatory ones. Its cytoprotective effect in retinal pigment epithelium cells under high glucose conditions makes it a potential support in the treatment of diabetic retinopathy. Despite its benefits, poor solubility in water reduces its potential for therapeutic use, making it the biggest biopharmaceutical challenge. The design of diosmin-loaded nanocarriers for topical ophthalmic application represents a novelty that has not been yet explored. For this purpose, the response surface methodology (RSM) was used to optimize nanostructured lipid carriers (NLCs), compatible for ocular administration, to encapsulate diosmin and improve its physicochemical issues. NLCs were prepared by a simple and scalable technique: a melt emulsification method followed by ultrasonication. The experimental design was composed of four independent variables (solid lipid concentration, liquid lipid concentration, surfactant concentration and type of solid lipid). The effect of the factors was assessed on NLC size and PDI (responses) by analysis of variance (ANOVA). The optimized formulation was selected according to the desirability function (0.993). Diosmin at two different concentrations (80 and 160 µM) was encapsulated into NLCs. Drug-loaded nanocarriers (D-NLCs) were subjected to a physicochemical and technological investigation revealing a mean particle size of 83.58 ± 0.77 nm and 82.21 ± 1.12 nm, respectively for the D-NLC formulation prepared with diosmin at the concentration of 80 µM or 160 µM, and a net negative surface charge (-18.5 ± 0.60 and -18.0 ± 1.18, respectively for the two batches). The formulations were analyzed in terms of pH (6.5), viscosity, and adjusted for osmolarity, making them more compatible with the ocular environment. Subsequently, stability studies were carried out to assess D-NLC behavior under different storage conditions up to 60 days, indicating a good stability of NLC samples at room temperature. In-vitro studies on ARPE-19 cells confirmed the cytocompatibility of NLCs with retinal epithelium. The effect of D-NLCs was also evaluated in-vitro on a model of retinal inflammation, demonstrating the cytoprotective effect of D-NLCs at various concentrations. RSM was found to be a reliable model to optimize NLCs for diosmin encapsulation.

Beneficial Effects of Standardized Extracts from Wastes of Red Oranges and Olive Leaves.

The awareness of the large amount of waste produced along the food chain, starting in the agricultural sector and continuing across industrial transformation to the domestic context, has in recent years also aroused strong concern amongst the public, who are ing about the possible consequences that this could have on environmental sustainability, resource waste and human health. The aim of the present research is the recovery of substances with high added value from waste and by-products typical of the Mediterranean area, such as the residue from the industrial processing of red oranges, called pastazzo (peels, pulps and seeds), which is particularly rich in anthocyanins, flavanones and hydroxycinnamic acids, and has numerous nutraceutical properties, as well as the olive leaves coming from olive-tree pruning, which are rich in substances such as oleuropein, elenolic acid, hydroxytyrosol, tyrosol and rutin. The effect of Red Orange Extract (ROE) and Olive Leaf Extract (OLE) on HepG2 fatty storage capacity was assessed performing Oil Red O' staining, and antioxidant properties of the extracts were evaluated following the steatosis model onset. Based on the results obtained, the preparation of natural extracts that are derived from these waste products can be useful for preventing, counteracting or delaying the onset of the complications of fatty liver disease, such as hepatic steatosis.

Novel Tyrosine Kinase Inhibitors to Target Chronic Myeloid Leukemia.

This paper reports on a novel series of tyrosine kinase inhibitors (TKIs) potentially useful for the treatment of chronic myeloid leukemia (CML). The newly designed and synthesized compounds are structurally related to nilotinib (NIL), a second-generation oral TKI, and to a series of imatinib (IM)-based TKIs, previously reported by our research group, these latter characterized by a hybrid structure between TKIs and heme oxygenase-1 (HO-1) inhibitors. The enzyme HO-1 was selected as an additional target since it is overexpressed in many cases of drug resistance, including CML. The new derivatives 1a-j correctly tackle the chimeric protein BCR-ABL. Therefore, the inhibition of TK was comparable to or higher than NIL and IM for many novel compounds, while most of the new analogs showed only moderate potency against HO-1. Molecular docking studies revealed insights into the binding mode with BCR-ABL and HO-1, providing a structural explanation for the differential activity. Cytotoxicity on K562 CML cells, both NIL-sensitive and -resistant, was evaluated. Notably, some new compounds strongly reduced the viability of K562 sensitive cells.

Heme Oxygenase Modulation Drives Ferroptosis in TNBC Cells.

The term ferroptosis refers to a peculiar type of programmed cell death (PCD) mainly characterized by extensive iron-dependent lipid peroxidation. Recently, ferroptosis has been suggested as a potential new strategy for the treatment of several cancers, including breast cancer (BC). In particular, among the BC subtypes, triple negative breast cancer (TNBC) is considered the most aggressive, and conventional drugs fail to provide long-term efficacy. In this context, our study's purpose was to investigate the mechanism of ferroptosis in breast cancer cell lines and reveal the significance of heme oxygenase (HO) modulation in the process, providing new biochemical approaches. HO's effect on BC was evaluated by MTT tests, gene silencing, Western blot analysis, and measurement of reactive oxygen species (ROS), glutathione (GSH) and lipid hydroperoxide (LOOH) levels. In order to assess HO's implication, different approaches were exploited, using two distinct HO-1 inducers (hemin and curcumin), a well-known HO inhibitor (SnMP) and a selective HO-2 inhibitor. The data obtained showed HO's contribution to the onset of ferroptosis; in particular, HO-1 induction seemed to accelerate the process. Moreover, our results suggest a potential role of HO-2 in erastin-induced ferroptosis. In view of the above, HO modulation in ferroptosis can offer a novel approach for breast cancer treatment.

Nanomedicine Strategies for Management of Drug Resistance in Lung Cancer.

Lung cancer (LC) is one of the leading causes of cancer occurrence and mortality worldwide. Treatment of patients with advanced and metastatic LC presents a significant challenge, as malignant cells use different mechanisms to resist chemotherapy. Drug resistance (DR) is a complex process that occurs due to a variety of genetic and acquired factors. Identifying the mechanisms underlying DR in LC patients and possible therapeutic alternatives for more efficient therapy is a central goal of LC research. Advances in nanotechnology resulted in the development of targeted and multifunctional nanoscale drug constructs. The possible modulation of the components of nanomedicine, their surface functionalization, and the encapsulation of various active therapeutics provide promising tools to bypass crucial biological barriers. These attributes enhance the delivery of multiple therapeutic agents directly to the tumor microenvironment (TME), resulting in reversal of LC resistance to anticancer treatment. This review provides a broad framework for understanding the different molecular mechanisms of DR in lung cancer, presents novel nanomedicine therapeutics aimed at improving the efficacy of treatment of various forms of resistant LC; outlines current challenges in using nanotechnology for reversing DR; and discusses the future directions for the clinical application of nanomedicine in the management of LC resistance.

Discovery of Novel Acetamide-Based Heme Oxygenase-1 Inhibitors with Potent In Vitro Antiproliferative Activity.

Heme oxygenase-1 (HO-1) promotes heme catabolism exercising cytoprotective roles in normal and cancer cells. Herein, we report the design, synthesis, molecular modeling, and biological evaluation of novel HO-1 inhibitors. Specifically, an amide linker in the central spacer and an imidazole were fixed, and the hydrophobic moiety required by the pharmacophore was largely modified. In many tumors, overexpression of HO-1 correlates with poor prognosis and chemoresistance, suggesting the inhibition of HO-1 as a possible antitumor strategy. Accordingly, compounds 7i and 7l-p emerged for their potency against HO-1 and were investigated for their anticancer activity against prostate (DU145), lung (A549), and glioblastoma (U87MG, A172) cancer cells. The selected compounds showed the best activity toward U87MG cells. Compound 7l was further investigated for its in-cell enzymatic HO-1 activity, expression levels, and effects on cell invasion and vascular endothelial growth factor (VEGF) extracellular release. The obtained data suggest that 7l can reduce cell invasivity acting through modulation of HO-1 expression.

Bioactive Compounds from Lemon (Citrus limon) Extract Overcome TNF-α-Induced Insulin Resistance in Cultured Adipocytes.

The consumption of plant-based food is important for health promotion, especially regarding the prevention and management of chronic diseases such as diabetes. We investigated the effects of a lemon extract (LE), containing ≥20.0% total flavanones and ≥1.0% total hydroxycinnamic acids, on insulin signaling in murine 3T3-L1 adipocytes treated with TNF-α, which was used to mimic in vitro the insulin resistance condition that characterizes diabetes mellitus. Our results showed LE increased PPARγ, GLUT4 and DGAT-1 levels, demonstrating the potential of this lemon extract in the management of insulin resistance conditions associated with TNF-α pathway activation. LE treatment further decreased the release of interleukin 6 (IL-6) and restored triglyceride synthesis, which is the main feature of a healthy adipocyte.

Tin Mesoporphyrin Selectively Reduces Non-Small-Cell Lung Cancer Cell Line A549 Proliferation by Interfering with Heme Oxygenase and Glutathione Systems.

In order to maintain redox homeostasis, non-small-cell lung cancer (NSCLC) increases the activation of many antioxidant systems, including the heme-oxygenase (HO) system. The overexpression of HO-1 has been often associated with chemoresistance and tumor aggressiveness. Our results clearly showed an overexpression of the HO-1 protein in A549 NSCLC cell lines compared to that in non-cancerous cells. Thus, we hypothesized that "off-label" use of tin mesoporphyrin, a well-known HO activity inhibitor clinically used for neonatal hyperbilirubinemia, has potential use as an anti-cancer agent. The pharmacological inhibition of HO activity caused a reduction in cell proliferation and migration of A549. SnMP treatment caused an increase in oxidative stress, as demonstrated by the upregulation of reactive oxygen species (ROS) and the depletion of glutathione (GSH) content. To support these data, Western blot analysis was performed to analyze glucose-6-phosphate dehydrogenase (G6PD), TP53-induced glycolysis and the apoptosis regulator (TIGAR), and the glutamate cysteine ligase catalytic (GCLC) subunit, as they represent the main regulators of the pentose phosphate pathway (PPP) and glutathione synthesis, respectively. NCI-H292, a subtype of the NSCLC cell line, did not respond to SnMP treatment, possibly due to low basal levels of HO-1, suggesting a cellular-dependent antitumorigenic effect. Altogether, our results suggest HO activity inhibition may represent a potential target for selective chemotherapy in lung cancer subtypes.

Novel mutual prodrug of 5-fluorouracil and heme oxygenase-1 inhibitor (5-FU/HO-1 hybrid): design and preliminary in vitro evaluation.

In this work, the first mutual prodrug of 5-fluorouracil and heme oxygenase1 inhibitor (5-FU/HO-1 hybrid) has been designed, synthesised, and evaluated for its in vitro chemical and enzymatic hydrolysis stability. Predicted in silico physicochemical properties of the newly synthesised hybrid (3) demonstrated a drug-like profile with suitable Absorption, Distribution, Metabolism, and Excretion (ADME) properties and low toxic liabilities. Preliminary cytotoxicity evaluation towards human prostate (DU145) and lung (A549) cancer cell lines demonstrated that 3 exerted a similar effect on cell viability to that produced by the reference drug 5-FU. Among the two tested cancer cell lines, the A549 cells were more susceptible for 3. Of note, hybrid 3 also had a significantly lower cytotoxic effect on healthy human lung epithelial cells (BEAS-2B) than 5-FU. Altogether our results served as an initial proof-of-concept to develop 5-FU/HO-1 mutual prodrugs as potential novel anticancer agents.

Heme Oxygenase-1 Signaling and Redox Homeostasis in Physiopathological Conditions.

Heme-oxygenase is the enzyme responsible for degradation of endogenous iron protoporphyirin heme; it catalyzes the reaction's rate-limiting step, resulting in the release of carbon monoxide (CO), ferrous ions, and biliverdin (BV), which is successively reduced in bilirubin (BR) by biliverdin reductase. Several studies have drawn attention to the controversial role of HO-1, the enzyme inducible isoform, pointing out its implications in cancer and other diseases development, but also underlining the importance of its antioxidant activity. The contribution of HO-1 in redox homeostasis leads to a relevant decrease in cells oxidative damage, which can be reconducted to its cytoprotective effects explicated alongside other endogenous mechanisms involving genes like TIGAR (TP53-induced glycolysis and apoptosis regulator), but also to the therapeutic functions of heme main transformation products, especially carbon monoxide (CO), which has been shown to be effective on GSH levels implementation sustaining body's antioxidant response to oxidative stress. The aim of this review was to collect most of the knowledge on HO-1 from literature, analyzing different perspectives to try and put forward a hypothesis on revealing yet unknown HO-1-involved pathways that could be useful to promote development of new therapeutical strategies, and lay the foundation for further investigation to fully understand this important antioxidant system.