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.

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.

Intravascular hemolysis leads to exaggerated corpus cavernosum relaxation: Implication for priapism in sickle cell disease.

Patients with sickle cell disease (SCD) display priapism. Clinical studies have shown a strong positive correlation between priapism and high levels of intravascular hemolysis in men with SCD. However, there are no experimental studies that show that intravascular hemolysis promotes alterations in erectile function. Therefore, we aimed to evaluate the corpus cavernosum smooth muscle relaxant function in a murine model that displays intravascular hemolysis induced by phenylhydrazine (PHZ), as well as the role of intravascular hemolysis in increasing the stress oxidative in the penis. Corpus cavernosum strips were dissected free and placed in organ baths. Acetylcholine and electrical field stimulation (EFS)-induced corpus cavernosum relaxations in vitro were obtained. Increased corpus cavernosum relaxant responses to acetylcholine and EFS were observed in the PHZ group. Protein expression of heme oxygenase-1 increased in the corpus cavernosum of the PHZ group, but PDE5 protein expression was not modified. Preincubation with the heme oxygenase inhibitor 1 J completely reversed the increased relaxant responses to acetylcholine and EFS in PHZ mice. Protein expression of NADPH oxidase subunit gp91phox, 3-nitrotyrosine, and 4-hydroxynonenal increased in the corpus cavernosum of the PHZ group, suggesting a state of oxidative stress. Basal cGMP production was lower in the PHZ group. Our results show that intravascular hemolysis promotes increased corpus cavernosum smooth muscle relaxation associated with increased HO-1 expression, as well as increased oxidative stress associated with upregulation of gp91phox expression. Moreover, our study supports clinical studies that point to a strong positive correlation between priapism and high levels of intravascular hemolysis in men with SCD.

Structure-activity relationships of mixed σ1R/σ2R ligands with antiproliferative and anticancer effects.

The overexpression of σ receptors (σRs) in various types of tumors has prompted a deep investigation of their role in cancer pathophysiology. Consequently, σR ligands have been widely studied in vitro and in vivo for their antiproliferative effects as a novel potential class of chemotherapeutic agents, both alone and in combination with other anticancer drugs. A growing body of evidence highlights that σR ligands can inhibit cancer cells' survival, migration, and proliferation, thanks to the modulation of a wide panel of tumorigenic pathways. In addition to their antitumor activity, σR ligands are gaining momentum as radiotracers for PET and SPECT imaging applications. The purpose of this review is to report on recent advances in the development of σR ligands. In particular, herein, we describe the structure-activity relationships of structurally diverse mixed σ1R/σ2R ligands that showed promising antitumor profiles towards a variety of cancer cell lines.

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.

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.

In Vivo Evaluation of Anti-Nociceptive Effects of Silver Nanoparticles.

Silver nanoparticles (AgNPs) are widely used commercially due to their antimicrobial effects. Little is known about the effect of AgNPs on neural transmission and pain response. The aim of this study was to assess the anti-nociceptive activity of AgNPs. AgNPs were prepared at 16 ug/mL, white albino rats were injected with various doses of AgNPs, and challenged using a hot-plate test and paw withdrawal latency (PWL) was measured. The chronic constriction injury (CCI) model was utilized to evaluate the pedal withdrawal reflex and tail withdrawal reflex. An electrophysiological study was conducted utilizing colon longitudinal muscle strips. AgNPs increased the latency of PWL in a dose-dependent matter over the duration of 6 h. The paw withdrawal threshold in animals with CCI significantly increased after AgNPs administration. In isolated colon longitudinal muscle strips, AgNPs significantly reduced the colonic migrating motor complexes (MMCs) and contraction. This action was completely reversed after removing the AgNPs and adding acetylcholine to the preparation. In this study, AgNPs showed significant anti-nociception properties. To our knowledge, this is the first report to describe this pharmacological action of AgNPs.

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.

A computer-aided approach to identify novel Leishmania major protein disulfide isomerase inhibitors for treatment of leishmaniasis.

Leishmaniasis is an infectious disease caused by parasites of the genus Leishmania and transmitted by the bite of a sand fly. To date, most available drugs for treatment are toxic and beyond the economic means of those affected by the disease. Protein disulfide isomerase (PDI) is a chaperone protein that plays a major role in the folding of newly synthesized proteins, specifically assisting in disulfide bond formation, breakage, or rearrangement in all non-native proteins. In previous work, we demonstrated that Leishmania major PDI (LmPDI) has an essential role in pathogen virulence. Furthermore, inhibition of LmPDI further blocked parasite infection in macrophages. In this study, we utilized a computer-aided approach to design a series of LmPDI inhibitors. Fragment-based virtual screening allowed for the understanding of the inhibitors' modes of action on LmPDI active sites. The generated compounds obtained after multiple rounds of virtual screening were synthesized and significantly inhibited target LmPDI reductase activity and were shown to decrease in vitro parasite growth in human monocyte-derived macrophages. This novel cheminformatics and synthetic approach led to the identification of a new series of compounds that might be optimized into novel drugs, likely more specific and less toxic for the treatment of leishmaniasis.

Growing the molecular architecture of imidazole-like ligands in HO-1 complexes.

Up-regulation of HO-1 had been frequently reported in different cases and types of human malignancies. Since poor clinical outcomes are reported in these cases, this enzyme's inhibition is considered a valuable and proven anticancer approach. To identify novel HO-1 inhibitors suitable for drug development, we report a structure-guided fragment-based approach to identify new lead compounds. Different parts of the selected molecules were analyzed, and the different series of novel compounds were virtually evaluated. The growing experiments of the classical HO-1 inhibitors structure led us to different hit-compounds. A synthetic pathway for six selected molecules was designed, and the compounds were synthesized. The biological activity revealed that molecules 10 and 12 inhibit the HO-1 activity with an IC50 of 1.01 and 0.90 µM, respectively. This study suggested that our growing approach was successful, and these results are ongoing for further development.

Selective Targeting of Breast Cancer by Tafuramycin A Using SMA-Nanoassemblies.

Triple-negative breast cancer (TNBC) is a heterogeneous subtype of tumors that tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein. The mainstay of treatment remains chemotherapy, but the therapeutic outcome remains inadequate. This paper investigates the potential of a duocarmycin derivative, tafuramycin A (TFA), as a new and more effective chemotherapy agent in TNBC treatment. To this extent, we optimized the chemical synthesis of TFA, and we encapsulated TFA in a micellar system to reduce side effects and increase tumor accumulation. In vitro and in vivo studies suggest that both TFA and SMA-TFA possess high anticancer effects in TNBC models. Finally, the encapsulation of TFA offered a preferential avenue to tumor accumulation by increasing its concentration at the tumor tissues by around four times in comparison with the free drug. Overall, the results provide a new potential strategy useful for TNBC treatment.

Non-competitive heme oxygenase-1 activity inhibitor reduces non-small cell lung cancer glutathione content and regulates cell proliferation.

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.

Identification of a potent heme oxygenase-2 (HO-2) inhibitor by targeting the secondary hydrophobic pocket of the HO-2 western region.

The enzymatic family of heme oxygenase (HO) is accountable for heme breakdown. Among the two isoforms characterized to date, HO-2 is poorly investigated due to the lack of potent HO-2 chemical modulators and the greater attentiveness towards HO-1 isoform. In the present paper, we report the rational design and synthesis of HO-2 inhibitors achieved by modulating the volume of known HO-1 inhibitors. The inhibition preference has been moved from HO-1 to HO-2 by merely increasing the volume of the substituent in the western region of the inhibitors. Docking studies demonstrated that new derivatives soak differently in the two binding pockets, probably due to the presence of a Tyr187 residue in HO-2. These findings could be useful for the design of new selective HO-2 compounds.

Chromatograpic resolution of phenylethanolic-azole racemic compounds highlighted stereoselective inhibition of heme oxygenase-1 by (R)-enantiomers.

Heme oxygenase-1 (HO-1) has been recognized as extensively involved in the development and aggravation of cancer, cell propagation and at in the mechanism of chemoresistance development. Low micromolar HO-1 inhibitors selective towards HO-2 has been recently reported, wherein the azole core and the hydrophobic residues are linked through a phenylethanolic spacer bearing a chiral center. Since less information are known about the stereoselective requirements for HO-1 inhibition, here we report the enantiomeric resolution of 1-(biphenyl-3-yl)-2-(1H-imidazol-1-yl)ethanol (1) and 1-[4-[(4-bromobenzyl)oxy]phenyl]-2-(1H-imidazol-1-yl)ethanol (2), two among the most potent and selective HO-1 inhibitors known thus far when tested as racemates. The absolute configuration was established for 1 by a combination of experimental and in silico derived electronic circular dichroism spectra, while docking approaches were useful in the case of compound 2. Biological evaluation of pure enantiomers highlighted higher HO-1 inhibitory activity of (R)-enantiomers. Docking studies demonstrated the importance of hydrogen bond interaction, more pronounced for the (R)-enantiomers, with a consensus water molecule within the binding pocket. The present study demonstrates that differences in three-dimensional structure amongst compounds 1 and 2 enantiomers affect significantly the selectivity of these HO-1 inhibitors.

Novel Heme Oxygenase-1 (HO-1) Inducers Based on Dimethyl Fumarate Structure.

Novel heme oxygenase-1 (HO-1) inducers based on dimethyl fumarate (DMF) structure are reported in this paper. These compounds are obtained by modification of the DMF backbone. Particularly, maintaining the α, ß-unsaturated dicarbonyl function as the central chain crucial for HO-1 induction, different substituted or unsubstituted phenyl rings are introduced by means of an ester or amide linkage. Symmetric and asymmetric derivatives are synthesized. All compounds are tested on a human hepatic stellate cell line LX-2 to assay their capacity for modifying HO-1 expression. Compounds 1b, 1l and 1m stand out for their potency as HO-1 inducers, being 2-3 fold more active than DMF, and for their ability to reverse reactive oxygen species (ROS) production mediated using palmitic acid (PA). These properties, coupled with a low toxicity toward LX-2 cell lines, make these compounds potentially useful for treatment of diseases in which HO-1 overexpression may counteract inflammation, such as hepatic fibrosis. Docking studies show a correlation between predicted binding free energy and experimental HO-1 expression data. These preliminary results may support the development of new approaches in the management of liver fibrosis.

New Arylethanolimidazole Derivatives as HO-1 Inhibitors with Cytotoxicity against MCF-7 Breast Cancer Cells.

In this paper, a novel series of imidazole-based heme oxygenase-1 (HO-1) inhibitors is reported. These compounds were obtained by modifications of previously described high potent and selective arylethanolimidazoles. In particular, simplification of the central linker and repositioning of the hydrophobic portion were carried out. Results indicate that a hydroxyl group in the central region is crucial for the potency as well as the spatial distribution of the hydrophobic portion. Docking studies revealed a similar interaction of the classical HO-1 inhibitors with the active site of the protein. The most potent and selective compound (5a) was tested for its potential cytotoxic activity against hormone-sensitive and hormone-resistant breast cancer cell lines (MCF-7 and MDA-MB-231).

Synthesis and Molecular Modelling Studies of New 1,3-Diaryl-5-Oxo-Proline Derivatives as Endothelin Receptor Ligands.

The synthesis of seventeen new 1,3-diaryl-5-oxo-proline derivatives as endothelin receptor (ETR) ligands is described. The structural configuration of the new molecules was determined by analyzing selected signals in proton NMR spectra. In vitro binding assays of the human ETA and ETB receptors allowed us to identify compound 31h as a selective ETAR ligand. The molecular docking of the selected compounds and the ETA antagonist atrasentan in the ETAR homology model provided insight into the structural elements required for the affinity and the selectivity of the ETAR subtype.

Evaluation of the status quo of polyphenols analysis: Part II-Analysis methods and food processing effects.

Nowadays due to the concern with the environmental impact of analytical techniques and in order to reduce the ecological footprint there is a tendency to use more efficient and faster procedures that use a smaller amount of organic solvents. Polyphenols have been widely studied in plant-based matrices due to their wide and potent biological properties; however there are no standardized procedures both for sample preparation and analysis of these compounds. The second of a two-part review will carry out a critical review of the extraction procedures and analytical methods applied to polyphenols and their selection criteria over a wide range of factors in relation to commerce-associated, environmental, and economic factors. It is foreseen that in the future the analysis of polyphenols in plant-based matrices includes the use of techniques that allow the simultaneous determination of different subclasses of polyphenols using fast, sophisticated, and automated techniques that allow the minimal consumption of solvents.

Targeting STATs in neuroinflammation: The road less traveled!

JAK/STAT transduction pathway is a highly conserved pathway implicated in regulating cellular proliferation, differentiation, survival and apoptosis. Dysregulation of this pathway is involved in the onset of autoimmune, haematological, oncological, metabolic and neurological diseases. Over the last few years, the research of anti-neuroinflammatory agents has gained considerable attention. The ability to diminish the STAT-induced transcription of inflammatory genes is documented for both natural compounds (such as polyphenols) and chemical drugs. Among polyphenols, quercetin and curcumin directly inhibit STAT, while Berberis vulgaris L. and Sophora alopecuroides L extracts act indirectly. Also, the Food and Drug Administration has approved several JAK/STAT inhibitors (direct or indirect) for treating inflammatory diseases, indicating STAT can be considered as a therapeutic target for neuroinflammatory pathologies. Considering the encouraging data obtained so far, clinical trials are warranted to demonstrate the effectiveness and potential use in the clinical practice of STAT inhibitors to treat inflammation-associated neurodegenerative pathologies.

Fourfold Filtered Statistical/Computational Approach for the Identification of Imidazole Compounds as HO-1 Inhibitors from Natural Products.

Over-regulation of Heme oxygenase 1 (HO-1) has been recently identified in many types of human cancer, and in these cases, poor clinical outcomes are normally reported. Indeed, the inhibition of HO-1 is being considered as an anticancer approach. Imidazole scaffold is normally present in most of the classical HO-1 inhibitors and seems indispensable to the inhibitory activity due to its strong interaction with the Fe(II) of the heme group. In this paper, we searched for new potentially HO-1 inhibitors among three different databases: Marine Natural Products (MNP), ZINC Natural Products (ZNP) and Super Natural II (SN2). 484,527 compounds were retrieved from the databases and filtered through four statistical/computational filters (2D descriptors, 2D-QSAR pharmacophoric model, 3D-QSAR pharmacophoric model, and docking). Different imidazole-based compounds were suggested by our methodology to be potentially active in inhibiting the HO-1, and the results have been rationalized by the bioactivity of the filtered molecules reported in the literature.