Daniela Rossin - Academia.edu (original) (raw)

Papers by Daniela Rossin

Biomedicines

Human mesenchymal stem cell (hMSC)-based therapy is an emerging resource in regenerative medicine... more Human mesenchymal stem cell (hMSC)-based therapy is an emerging resource in regenerative medicine. Despite the innate ability of hMSCs to migrate to sites of injury, homing of infused hMSCs to the target tissue is inefficient. It was shown that silica nanoparticles (SiO2-NPs), previously developed to track the stem cells after transplantation, accumulated in lysosomes leading to a transient blockage of the autophagic flux. Since CXCR4 turnover is mainly regulated by autophagy, we tested the effect of SiO2-NPs on chemotactic migration of hMSCs along the SDF1α/CXCR4 axis that plays a pivotal role in directing MSC homing to sites of injury. Our results showed that SiO2-NP internalization augmented CXCR4 surface levels. We demonstrated that SiO2-NP-dependent CXCR4 increase was transient, and it reversed at the same time as lysosomal compartment normalization. Furthermore, the autophagy inhibitor Bafilomycin-A1 reproduced CXCR4 overexpression in control hMSCs confirming the direct effect...

International Journal of Molecular Sciences

Myocardial infarction (MI) is one of the leading causes of heart-related deaths worldwide. Follow... more Myocardial infarction (MI) is one of the leading causes of heart-related deaths worldwide. Following MI, the hypoxic microenvironment triggers apoptosis, disrupts the extracellular matrix and forms a non-functional scar that leads towards adverse left ventricular (LV) remodelling. If left untreated this eventually leads to heart failure. Besides extensive advancement in medical therapy, complete functional recovery is never accomplished, as the heart possesses limited regenerative ability. In recent decades, the focus has shifted towards tissue engineering and regenerative strategies that provide an attractive option to improve cardiac regeneration, limit adverse LV remodelling and restore function in an infarcted heart. Acellular scaffolds possess attractive features that have made them a promising therapeutic candidate. Their application in infarcted areas has been shown to improve LV remodelling and enhance functional recovery in post-MI hearts. This review will summarise the upd...

Biomedicines, 2021

Chemotherapeutics represent the standard treatment for a wide range of cancers. However, these ag... more Chemotherapeutics represent the standard treatment for a wide range of cancers. However, these agents also affect healthy cells, thus leading to severe off-target effects. Given the non-selectivity of the commonly used drugs, any increase in the selective tumor tissue uptake would represent a significant improvement in cancer therapy. Recently, the use of gene therapy to completely remove the lesion and avoid the toxicity of chemotherapeutics has become a tendency in oncotherapy. Ideally, the genetic material must be safely transferred from the site of administration to the target cells, without involving healthy tissues. This can be achieved by encapsulating genes into non-viral carriers and modifying their surface with ligands with high selectivity and affinity for a relevant receptor on the target cells. Hence, in this work we evaluate the use of terpolymer-based nanocapsules for the targeted delivery of DNA toward cancer cells. The surface of the nanocapsules is decorated with f...

Antioxidants

Background: Cocoa bean shell (CBS), a main byproduct of cocoa processing, represents a source of ... more Background: Cocoa bean shell (CBS), a main byproduct of cocoa processing, represents a source of components such as polyphenols and methylxanthines, which have been associated with a reduced risk of several diseases. Therefore, CBS has potential application as a food ingredient. Intestinal mucosa is exposed to immune and inflammatory responses triggered by dietary agents, such as oxysterols, which derive from cholesterol oxidation and are pro-oxidant compounds able to affect intestinal function. We aimed at investigating the capability of the Forastero cultivar CBS, added or not added to ice cream, to protect against the intestinal barrier damage induced by a dietary oxysterol mixture. Methods: Composition and antioxidant capacity of in vitro digested CBS and CBS-enriched ice cream were analyzed by high-performance liquid chromatography and 1,1-diphenyl-2-picryl-hydrazyl radical-scavenging assay, respectively. CaCo-2 cells differentiated into enterocyte-like monolayer were incubated...

Archives of Biochemistry and Biophysics

The alteration of the intestinal barrier function is currently believed to be involved in the pat... more The alteration of the intestinal barrier function is currently believed to be involved in the pathogenesis of gut diseases mainly associated with the activation of inflammation processes. Diet plays an important role in the control of human gut integrity. Theobromine is a natural methylxanthine present in dark chocolate particularly abundant in cocoa bean shell. This is a polyphenol rich by-product generated in cocoa industrial processing, which is gaining value as a functional ingredient. This study aims to highlight for the first time the capability of theobromine in protecting the intestinal cell monolayer from a mixture of dietary oxysterols showing an inflammatory action in terms of IL-8 and MCP-1 overproduction. Differentiated CaCo-2 cells were treated with 60 μM oxysterol mixture and pre-incubated with 10 μM theobromine. Intestinal barrier damage was investigated in terms of tight junction claudin 1, occludin and JAM-A protein levels, matrix metalloproteinase (MMP) -2 and -9 activation and anti/pro-apoptotic protein changes. The observed cell monolayer permeability protection by theobromine may be due to its ability to inhibit the production of cytokines and MMPs that can be responsible for tight junction loss and apoptosis in intestinal cells. Our findings provide additional mechanistic hints on the healthy effect of theobromine cocoa component as an attractive natural molecule in the prevention of inflammatory gut diseases.

Cells

Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Mitotane, the standard treat... more Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Mitotane, the standard treatment for ACC, impairs adrenocortical steroid biosynthesis and cholesterol metabolism. In the H295R cell line, a standard ACC in vitro model, mitotane was previously reported to enhance the production of some oxysterols. To verify the possible mechanistic involvement of oxysterols in the anti-ACC effect of mitotane, a gas chromatography mass spectrometry (GC-MS) profiling of oxysterols and the main cholesterol precursors was carried out in H295R cells. Among the oxysterols detected in mitotane-treated cells, 27OHC was markedly produced, as well as lanosterol and lathosterol cholesterol precursors. In this cell model, mitotane was confirmed to affect mitochondrial transmembrane potential and induce apoptosis. Such cytotoxic effects were perfectly matched by H295R cell treatment with a single identical micromolar amount of 27OHC. The mitotane-dependent strong increase in 27OHC was confirmed...

Free Radical Biology and Medicine

Abstract The emerging role of the diet in the incidence of intestinal inflammatory diseases has s... more Abstract The emerging role of the diet in the incidence of intestinal inflammatory diseases has stimulated research on the influence of eating habits with pro‐inflammatory properties in inducing epithelial barrier disturbance. Cholesterol oxidation products, namely oxysterols, have been shown to promote and sustain oxidative/inflammatory reactions in human digestive tract. This work investigated in an in vitro model the potential ability of a combination of dietary oxysterols representative of a hyper‐cholesterol diet to induce the loss of intestinal epithelial layer integrity. The components of the experimental mixture were the main oxysterols stemming from heat‐induced cholesterol auto‐oxidation, namely 7‐ketocholesterol, 5&agr;,6&agr;‐and 5&bgr;,6&bgr;‐epoxycholesterol, 7&agr;‐ and 7&bgr;‐hydroxycholesterol. These compounds added to monolayers of differentiated CaCo‐2 cells in combination or singularly, caused a time‐dependent induction of matrix metalloproteinases (MMP)‐2 and ‐9, also known as gelatinases. The hyperactivation of MMP‐2 and ‐9 was found to be associated with decreased levels of the tight junctions zonula occludens‐1 (ZO‐1), occludin and Junction Adhesion Molecule‐A (JAM‐A). Together with such a protein loss, particularly evident for ZO‐1, a net perturbation of spatial localization of the three tight junctions was observed. Cell monolayer pre‐treatment with the selective inhibitor of MMPs ARP100 or polyphenol (‐)‐epicathechin, previously shown to inhibit NADPH oxidase in the same model system, demonstrated that the decrease of the three tight junction proteins was mainly a consequence of MMPs induction, which was in turn dependent on the pro‐oxidant property of the oxysterols investigated. Although further investigation on oxysterols intestinal layer damage mechanism is to be carried on, the consequent ‐ but incomplete ‐ prevention of oxysterols‐dependent TJs alteration due to MMPs inhibition, avoided the loss of scaffold protein ZO‐1, with possible significant recovery of intestinal monolayer integrity. Graphical abstract No caption available. HighlightsOxysterols produced in cholesterol‐rich foods alter intestinal epithelial layer.Matrix metalloproteinases (MMPs)‐2 and ‐9 are activated by dietary oxysterols.Oxysterols affect MMP‐2 and ‐9 when used in combination as they are in food.Oxy‐mix disrupts intestinal tight junctions mainly through MMP‐2 and‐9 induction.These effects depend on the pro‐oxidant/inflammatory properties of dietary oxysterols.

Antioxidants

Background: Exaggerated Toll-like receptor (TLR)-mediated immune and inflammatory responses play ... more Background: Exaggerated Toll-like receptor (TLR)-mediated immune and inflammatory responses play a role in inflammatory bowel diseases. This report deals with the ability of a mixture of oxysterols widely present in cholesterol-rich foods to induce in vitro intestinal inflammation through TLR up-regulation. The anti-inflammatory action of four cocoa bean shell (CBS) extracts with different polyphenol content, was tested. Methods: Differentiated intestinal CaCo-2 cells were treated with a dietary oxysterol mixture (Oxy-mix) (60 µM). The expression and activation of TLR2 and TLR4, as well as the production of their downstream signaling effectors IL-8, IFNβ and TNFα were analyzed in the presence or absence of TLR antibodies. Honduras CBS extracts were characterized for their polyphenol contents; their anti-inflammatory action was analyzed in CaCo-2 cells treated with Oxy-mix. Results: Oxysterol-dependent TLR-2 and TLR4 over-expression and activation together with cytokine induction wer...

Free Radical Biology and Medicine

High amounts of cholesterol have been definitely associated with the pathogenesis of several dise... more High amounts of cholesterol have been definitely associated with the pathogenesis of several diseases, including metabolic and neurodegenerative disorders, cardiovascular diseases, and cancer. In all these pathologies the exacerbation of pro-oxidant and inflammatory responses is a consistent feature. In this scenario, species derived from enzymatic and non-enzymatic cholesterol oxidation, namely oxysterols, are strongly suspected to play a primary role. The consideration of these bioactive lipids is therefore helpful in investigating pathological mechanisms and may also acquire clinical value for the diagnosis and treatment of the disease. For this purpose and considering that a great number of oxysterols may be present together in the body, the employment of lipidomics technology certainly represents a powerful strategy for the simultaneous detection and characterization of these compounds in biological specimens. In this review we will discuss the applicability of lipidomics approach in the study of the association between oxysterols and diseases.

Free Radical Biology and Medicine

Increased production of 27-hydroxycholesterol in human colorectal cancer advanced stage: Possible... more Increased production of 27-hydroxycholesterol in human colorectal cancer advanced stage: Possible contribution to cancer cell survival and infiltration

Biochimie

Вступ. Стрімкі сучасні реформувальні процеси охопили усі галузі українського суспільства. Медична... more Вступ. Стрімкі сучасні реформувальні процеси охопили усі галузі українського суспільства. Медична освіта не стала винятком. Особлива увага звертається до молодих фахівців, здатних швидко обробляти масиви змінювальної інформації, критично аналізувати, легко мобілізуватися, а відтак, бути конкурентоспроможним на ринку праці. Тож, під час навчання у вищому навчальному закладі, значення набуває самостійна робота студентів, правильна організація якої забезпечить становлення таких особистісних якостей, як саморозвиток, самовдосконалення, в ідповідальність за якість власних знань. Не останню роль відіграє й значне зменшення навчальними планами часу, відведеного на теоретичні заняття. Тож, в умовах аудиторії можуть розглядатись та засвоюватись лише ті навчальні фрейми, які, з огляду на обсяг, наявність суперечливих моментів, неоднозначність трактування або необхідність безпосереднього контролю викладача не можуть бути засвоєні самостійно. У такому ракурсі, значення набувають зміст, мета, види, форми, методи, обсяг, з'ясування часових витрат, формулювання завдань та прийоми контролю.

Redox Biology

Dietary habits may strongly influence intestinal homeostasis. Oxysterols, the oxidized products o... more Dietary habits may strongly influence intestinal homeostasis. Oxysterols, the oxidized products of cholesterol present in cholesterol-containing foodstuffs, have been shown to exert pro-oxidant and pro-inflammatory effects, altering intestinal epithelial layer and thus contributing to the pathogenesis of human inflammatory bowel diseases and colon cancer. Extra virgin olive oil polyphenols possess antioxidant and anti-inflammatory properties, and concentrate in the intestinal lumen, where may help in preventing intestinal diseases. In the present study we evaluated the ability of an extra virgin olive oil phenolic extract to counteract the pro-oxidant and pro-inflammatory action of a representative mixture of dietary oxysterols in the human colon adenocarcinoma cell line (Caco-2) undergoing full differentiation into enterocyte-like cells. Oxysterols treatment significantly altered differentiated Caco-2 cells redox status, leading to oxidant species production and a decrease of GSH levels, after 1 h exposure, followed by an increase of cytokines production, IL-6 and IL-8, after 24 h. Oxysterol cell treatment also induced after 48 h an increase of NO release, due to the induction of iNOS. Pretreatment with the phenolic extract counteracted oxysterols effects, at least in part by modulating one of the main pathways activated in the cellular response to the action of oxysterols, the MAPK-NF-kB pathway. We demonstrated the ability of the phenolic extract to directly modulate p38 and JNK1/2 phosphorylation and activation of NF-kB, following its inhibitor IkB phosphorylation. The phenolic extract also inhibited iNOS induction, keeping NO concentration at the control level. Our results suggest a protective effect at intestinal level of extra virgin olive oil polyphenols, able to prevent or limit redox unbalance and the onset and progression of chronic intestinal inflammation.

Free Radical Biology and Medicine

Atherosclerosis is currently understood to be mainly the consequence of a complicated inflammator... more Atherosclerosis is currently understood to be mainly the consequence of a complicated inflammatory process at the different stages of plaque development. Among the several inflammatory molecules involved, up-regulation of the functional cyclooxygenase 2/membrane-bound prostaglandin E synthase 1 (COX-2/mPGES-1) axis plays a key role in plaque development. Excessive production of oxidized lipids, following low-density lipoprotein (LDL) oxidation, is a characteristic feature of atherosclerosis. Among LDLs, the oxysterol 27-hydroxycholesterol (27-OH) and the aldehyde 4-hydroxynonenal (HNE) substantially accumulate in the atherosclerotic plaque, contributing to its progression and instability through a variety of processes. This study shows that 27-OH and HNE promote up-regulation of both the inducible enzymes COX-2 and mPGES-1, leading to increased production of prostaglandin (PG) E2 and inducible nitric oxide synthase, and the subsequent release of nitric oxide in human promonocytic U937 cells. The study also examined the potential involvement of the functionally coupled COX-2/mPGES-1 in enhancing the production of certain pro-inflammatory cytokines and of matrix metalloproteinase 9 by U937 cells. This enhancement is presumably due to the induction of PGE2 synthesis, as a result of the up-regulation of the COX-2/mPGES-1, stimulated by the two oxidized lipids, 27-OH and HNE. Induction of PGE2 synthesis might thus be a mechanism of plaque instability and eventual rupture, contributing to matrix metalloproteinase production by activated macrophages.

Free Radical Biology and Medicine

Colorectal cancer (CRC) is one of the most common tumors world wild. High cholesterol diet is con... more Colorectal cancer (CRC) is one of the most common tumors world wild. High cholesterol diet is considered a risk for CRC development. Cholesterol oxidation products, namely oxysterols, have been shown to have a role in human degenerative diseases, mainly for their ability to favor inflammatory reactions. Therefore, they could be involved in inducing intestinal inflammation, a process strongly associated to colorectal carcinogenesis. Dietary oxysterols could derange intestinal epithelial barrier by inducing the activation of metalloproteinases (MMPs) as well as the decrease of tight junctions (TJs), which are essential in mucosa barrier maintenance. Enterocyte-like CaCo-2 cells were treated with a mixture of oxysterols representative of a hyper-cholesterol diet. The time course study of MMPs activity (MMP-9 and MMP-2) showed their significant increase, reaching the maximum at 72 hours treatment. Moreover, the dietary oxysterols decreased the TJ production, in particular of junctional adhesion molecule (JAM), zonula occludens (ZO) and occludin. These two events appeared to be associated. In fact, cell pretreatment with specific MMPs inhibitors restored TJ levels. Therefore, dietary oxysterols could be actually implicated in tumor progression towards a more aggressive phenotype by inducing extracellular matrix destabilization and intestinal barrier disruption.

Free Radical Biology and Medicine

A still growing bulk of evidence underlies the multifaceted biochemical properties of oxysterols,... more A still growing bulk of evidence underlies the multifaceted biochemical properties of oxysterols, several of them of clear relevance to human pathophysiology. Taken up by cells through both vesicular and non vesicular ways or often generated intracellularly, oxysterols contribute to modulate the inflammatory response of a given tissue but also cell viability, metabolism and function. The signaling pathways and the transcription factors whose activation they can influence, often through redox-mediated reactions, are quite a number. Moreover, the outcome of the complex network of intracellular reactions promoted by oxysterols appears to be largely dependent upon specific features and dynamic conditions of the cellular and tissue environment but also on their actual concentration in the site of action. Here focus was given to their potential implication in the advanced progression of atherosclerosis, whosehypercholesterolemia is a recognized primary risk factor. A significant contribution to the possible rupture of atherosclerotic plaque could be provided by defined oxysterols, in particular 27-hydroxycholesterol. Indeed, the latter oxysterol was shown in a promonocytic cell line to amplify the inflammatory reaction and activate several pathways leading to a marked up-regulation of metalloproteases, the key enzymes involved in cap matrix degradation and weakening.

Current medicinal chemistry, Jan 19, 2017

A defective mucosal barrier function is the principal cause of the uncontrolled onset and progres... more A defective mucosal barrier function is the principal cause of the uncontrolled onset and progression of a number of human inflammatory gut diseases, most of which are characterized by chronic intermittent immune and inflammatory responses leading to structural intestinal damage, which can represent a potential risk for colorectal cancer development. During the active disease phase the production of pro-inflammatory cytokines and chemokines, and the induction of oxidative reactions by activated leukocytes and epithelial cells represent the main event in intestinal inflammation. Oxidative stress plays a key role in the development of intestinal damage. Indeed reactive oxygen species and their oxidized by-products regulate redox-sensitive signaling pathways and transcription factors, which sustain inflammation within the intestinal layer. Polyunsaturated fatty acids and cholesterol are the principal targets of oxidative modifications. These lipids, which are cell membrane constituents...

Redox biology, Aug 23, 2017

Oxysterols are known pleiotropic molecules whose antiviral action has been recently discovered. H... more Oxysterols are known pleiotropic molecules whose antiviral action has been recently discovered. Here reported is the activity of a panel of oxysterols against HSV-1 with the identification of a new mechanism of action. A marked antiviral activity not only of 25HC but also of 27HC against HSV-1 was observed either if the oxysterols were added before or after infection, suggesting an activity unrelated to the viral entry inhibition as proposed by previous literature. Therefore, the relation between the pro-inflammatory activity of oxysterols and the activation of NF-kB and IL-6 induced by HSV-1 in the host cell was investigated. Indeed, cell pre-incubation with oxysterols further potentiated IL-6 production as induced by HSV-1 infection with a consequent boost of the interleukin's total cell secretion. Further, a direct antiviral effect of IL-6 administration to HSV-1 infected cells was demonstrated, disclosing an additional mechanism of antiviral action by both 25HC and 27HC.

Free Radical Biology and Medicine, 2017

Consistent experimental data suggest the importance of inflammation-associated oxidative stress i... more Consistent experimental data suggest the importance of inflammation-associated oxidative stress in colorectal cancer (CRC) pathogenesis. Inflammatory bowel disease with chronic intestinal inflammation is now considered a precancerous condition. Oxidative stress is an essential feature of inflammation. Activation of redox-sensitive pro-inflammatory cell signals and inflammatory mediators concur to establish a protumoral environment. In this frame, lipid oxidation products, namely 4-hydroxynonenal and oxysterols, can be produced in big quantity so as to be able to exert their function as inducers of cell signaling pathways of proliferation and survival. Notably, an important source of these two compounds is represented by a high fat diet, which is undoubtedly a risk factor for inflammation and CRC development. Current evidence for the emerging implication of these two oxidized lipids in inflammation and CRC development is discussed in this review.

Biomedicines

Human mesenchymal stem cell (hMSC)-based therapy is an emerging resource in regenerative medicine... more Human mesenchymal stem cell (hMSC)-based therapy is an emerging resource in regenerative medicine. Despite the innate ability of hMSCs to migrate to sites of injury, homing of infused hMSCs to the target tissue is inefficient. It was shown that silica nanoparticles (SiO2-NPs), previously developed to track the stem cells after transplantation, accumulated in lysosomes leading to a transient blockage of the autophagic flux. Since CXCR4 turnover is mainly regulated by autophagy, we tested the effect of SiO2-NPs on chemotactic migration of hMSCs along the SDF1α/CXCR4 axis that plays a pivotal role in directing MSC homing to sites of injury. Our results showed that SiO2-NP internalization augmented CXCR4 surface levels. We demonstrated that SiO2-NP-dependent CXCR4 increase was transient, and it reversed at the same time as lysosomal compartment normalization. Furthermore, the autophagy inhibitor Bafilomycin-A1 reproduced CXCR4 overexpression in control hMSCs confirming the direct effect...

International Journal of Molecular Sciences

Myocardial infarction (MI) is one of the leading causes of heart-related deaths worldwide. Follow... more Myocardial infarction (MI) is one of the leading causes of heart-related deaths worldwide. Following MI, the hypoxic microenvironment triggers apoptosis, disrupts the extracellular matrix and forms a non-functional scar that leads towards adverse left ventricular (LV) remodelling. If left untreated this eventually leads to heart failure. Besides extensive advancement in medical therapy, complete functional recovery is never accomplished, as the heart possesses limited regenerative ability. In recent decades, the focus has shifted towards tissue engineering and regenerative strategies that provide an attractive option to improve cardiac regeneration, limit adverse LV remodelling and restore function in an infarcted heart. Acellular scaffolds possess attractive features that have made them a promising therapeutic candidate. Their application in infarcted areas has been shown to improve LV remodelling and enhance functional recovery in post-MI hearts. This review will summarise the upd...

Biomedicines, 2021

Chemotherapeutics represent the standard treatment for a wide range of cancers. However, these ag... more Chemotherapeutics represent the standard treatment for a wide range of cancers. However, these agents also affect healthy cells, thus leading to severe off-target effects. Given the non-selectivity of the commonly used drugs, any increase in the selective tumor tissue uptake would represent a significant improvement in cancer therapy. Recently, the use of gene therapy to completely remove the lesion and avoid the toxicity of chemotherapeutics has become a tendency in oncotherapy. Ideally, the genetic material must be safely transferred from the site of administration to the target cells, without involving healthy tissues. This can be achieved by encapsulating genes into non-viral carriers and modifying their surface with ligands with high selectivity and affinity for a relevant receptor on the target cells. Hence, in this work we evaluate the use of terpolymer-based nanocapsules for the targeted delivery of DNA toward cancer cells. The surface of the nanocapsules is decorated with f...

Antioxidants

Background: Cocoa bean shell (CBS), a main byproduct of cocoa processing, represents a source of ... more Background: Cocoa bean shell (CBS), a main byproduct of cocoa processing, represents a source of components such as polyphenols and methylxanthines, which have been associated with a reduced risk of several diseases. Therefore, CBS has potential application as a food ingredient. Intestinal mucosa is exposed to immune and inflammatory responses triggered by dietary agents, such as oxysterols, which derive from cholesterol oxidation and are pro-oxidant compounds able to affect intestinal function. We aimed at investigating the capability of the Forastero cultivar CBS, added or not added to ice cream, to protect against the intestinal barrier damage induced by a dietary oxysterol mixture. Methods: Composition and antioxidant capacity of in vitro digested CBS and CBS-enriched ice cream were analyzed by high-performance liquid chromatography and 1,1-diphenyl-2-picryl-hydrazyl radical-scavenging assay, respectively. CaCo-2 cells differentiated into enterocyte-like monolayer were incubated...

Archives of Biochemistry and Biophysics

The alteration of the intestinal barrier function is currently believed to be involved in the pat... more The alteration of the intestinal barrier function is currently believed to be involved in the pathogenesis of gut diseases mainly associated with the activation of inflammation processes. Diet plays an important role in the control of human gut integrity. Theobromine is a natural methylxanthine present in dark chocolate particularly abundant in cocoa bean shell. This is a polyphenol rich by-product generated in cocoa industrial processing, which is gaining value as a functional ingredient. This study aims to highlight for the first time the capability of theobromine in protecting the intestinal cell monolayer from a mixture of dietary oxysterols showing an inflammatory action in terms of IL-8 and MCP-1 overproduction. Differentiated CaCo-2 cells were treated with 60 μM oxysterol mixture and pre-incubated with 10 μM theobromine. Intestinal barrier damage was investigated in terms of tight junction claudin 1, occludin and JAM-A protein levels, matrix metalloproteinase (MMP) -2 and -9 activation and anti/pro-apoptotic protein changes. The observed cell monolayer permeability protection by theobromine may be due to its ability to inhibit the production of cytokines and MMPs that can be responsible for tight junction loss and apoptosis in intestinal cells. Our findings provide additional mechanistic hints on the healthy effect of theobromine cocoa component as an attractive natural molecule in the prevention of inflammatory gut diseases.

Cells

Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Mitotane, the standard treat... more Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Mitotane, the standard treatment for ACC, impairs adrenocortical steroid biosynthesis and cholesterol metabolism. In the H295R cell line, a standard ACC in vitro model, mitotane was previously reported to enhance the production of some oxysterols. To verify the possible mechanistic involvement of oxysterols in the anti-ACC effect of mitotane, a gas chromatography mass spectrometry (GC-MS) profiling of oxysterols and the main cholesterol precursors was carried out in H295R cells. Among the oxysterols detected in mitotane-treated cells, 27OHC was markedly produced, as well as lanosterol and lathosterol cholesterol precursors. In this cell model, mitotane was confirmed to affect mitochondrial transmembrane potential and induce apoptosis. Such cytotoxic effects were perfectly matched by H295R cell treatment with a single identical micromolar amount of 27OHC. The mitotane-dependent strong increase in 27OHC was confirmed...

Free Radical Biology and Medicine

Abstract The emerging role of the diet in the incidence of intestinal inflammatory diseases has s... more Abstract The emerging role of the diet in the incidence of intestinal inflammatory diseases has stimulated research on the influence of eating habits with pro‐inflammatory properties in inducing epithelial barrier disturbance. Cholesterol oxidation products, namely oxysterols, have been shown to promote and sustain oxidative/inflammatory reactions in human digestive tract. This work investigated in an in vitro model the potential ability of a combination of dietary oxysterols representative of a hyper‐cholesterol diet to induce the loss of intestinal epithelial layer integrity. The components of the experimental mixture were the main oxysterols stemming from heat‐induced cholesterol auto‐oxidation, namely 7‐ketocholesterol, 5&agr;,6&agr;‐and 5&bgr;,6&bgr;‐epoxycholesterol, 7&agr;‐ and 7&bgr;‐hydroxycholesterol. These compounds added to monolayers of differentiated CaCo‐2 cells in combination or singularly, caused a time‐dependent induction of matrix metalloproteinases (MMP)‐2 and ‐9, also known as gelatinases. The hyperactivation of MMP‐2 and ‐9 was found to be associated with decreased levels of the tight junctions zonula occludens‐1 (ZO‐1), occludin and Junction Adhesion Molecule‐A (JAM‐A). Together with such a protein loss, particularly evident for ZO‐1, a net perturbation of spatial localization of the three tight junctions was observed. Cell monolayer pre‐treatment with the selective inhibitor of MMPs ARP100 or polyphenol (‐)‐epicathechin, previously shown to inhibit NADPH oxidase in the same model system, demonstrated that the decrease of the three tight junction proteins was mainly a consequence of MMPs induction, which was in turn dependent on the pro‐oxidant property of the oxysterols investigated. Although further investigation on oxysterols intestinal layer damage mechanism is to be carried on, the consequent ‐ but incomplete ‐ prevention of oxysterols‐dependent TJs alteration due to MMPs inhibition, avoided the loss of scaffold protein ZO‐1, with possible significant recovery of intestinal monolayer integrity. Graphical abstract No caption available. HighlightsOxysterols produced in cholesterol‐rich foods alter intestinal epithelial layer.Matrix metalloproteinases (MMPs)‐2 and ‐9 are activated by dietary oxysterols.Oxysterols affect MMP‐2 and ‐9 when used in combination as they are in food.Oxy‐mix disrupts intestinal tight junctions mainly through MMP‐2 and‐9 induction.These effects depend on the pro‐oxidant/inflammatory properties of dietary oxysterols.

Antioxidants

Background: Exaggerated Toll-like receptor (TLR)-mediated immune and inflammatory responses play ... more Background: Exaggerated Toll-like receptor (TLR)-mediated immune and inflammatory responses play a role in inflammatory bowel diseases. This report deals with the ability of a mixture of oxysterols widely present in cholesterol-rich foods to induce in vitro intestinal inflammation through TLR up-regulation. The anti-inflammatory action of four cocoa bean shell (CBS) extracts with different polyphenol content, was tested. Methods: Differentiated intestinal CaCo-2 cells were treated with a dietary oxysterol mixture (Oxy-mix) (60 µM). The expression and activation of TLR2 and TLR4, as well as the production of their downstream signaling effectors IL-8, IFNβ and TNFα were analyzed in the presence or absence of TLR antibodies. Honduras CBS extracts were characterized for their polyphenol contents; their anti-inflammatory action was analyzed in CaCo-2 cells treated with Oxy-mix. Results: Oxysterol-dependent TLR-2 and TLR4 over-expression and activation together with cytokine induction wer...

Free Radical Biology and Medicine

High amounts of cholesterol have been definitely associated with the pathogenesis of several dise... more High amounts of cholesterol have been definitely associated with the pathogenesis of several diseases, including metabolic and neurodegenerative disorders, cardiovascular diseases, and cancer. In all these pathologies the exacerbation of pro-oxidant and inflammatory responses is a consistent feature. In this scenario, species derived from enzymatic and non-enzymatic cholesterol oxidation, namely oxysterols, are strongly suspected to play a primary role. The consideration of these bioactive lipids is therefore helpful in investigating pathological mechanisms and may also acquire clinical value for the diagnosis and treatment of the disease. For this purpose and considering that a great number of oxysterols may be present together in the body, the employment of lipidomics technology certainly represents a powerful strategy for the simultaneous detection and characterization of these compounds in biological specimens. In this review we will discuss the applicability of lipidomics approach in the study of the association between oxysterols and diseases.

Free Radical Biology and Medicine

Increased production of 27-hydroxycholesterol in human colorectal cancer advanced stage: Possible... more Increased production of 27-hydroxycholesterol in human colorectal cancer advanced stage: Possible contribution to cancer cell survival and infiltration

Biochimie

Вступ. Стрімкі сучасні реформувальні процеси охопили усі галузі українського суспільства. Медична... more Вступ. Стрімкі сучасні реформувальні процеси охопили усі галузі українського суспільства. Медична освіта не стала винятком. Особлива увага звертається до молодих фахівців, здатних швидко обробляти масиви змінювальної інформації, критично аналізувати, легко мобілізуватися, а відтак, бути конкурентоспроможним на ринку праці. Тож, під час навчання у вищому навчальному закладі, значення набуває самостійна робота студентів, правильна організація якої забезпечить становлення таких особистісних якостей, як саморозвиток, самовдосконалення, в ідповідальність за якість власних знань. Не останню роль відіграє й значне зменшення навчальними планами часу, відведеного на теоретичні заняття. Тож, в умовах аудиторії можуть розглядатись та засвоюватись лише ті навчальні фрейми, які, з огляду на обсяг, наявність суперечливих моментів, неоднозначність трактування або необхідність безпосереднього контролю викладача не можуть бути засвоєні самостійно. У такому ракурсі, значення набувають зміст, мета, види, форми, методи, обсяг, з'ясування часових витрат, формулювання завдань та прийоми контролю.

Redox Biology

Dietary habits may strongly influence intestinal homeostasis. Oxysterols, the oxidized products o... more Dietary habits may strongly influence intestinal homeostasis. Oxysterols, the oxidized products of cholesterol present in cholesterol-containing foodstuffs, have been shown to exert pro-oxidant and pro-inflammatory effects, altering intestinal epithelial layer and thus contributing to the pathogenesis of human inflammatory bowel diseases and colon cancer. Extra virgin olive oil polyphenols possess antioxidant and anti-inflammatory properties, and concentrate in the intestinal lumen, where may help in preventing intestinal diseases. In the present study we evaluated the ability of an extra virgin olive oil phenolic extract to counteract the pro-oxidant and pro-inflammatory action of a representative mixture of dietary oxysterols in the human colon adenocarcinoma cell line (Caco-2) undergoing full differentiation into enterocyte-like cells. Oxysterols treatment significantly altered differentiated Caco-2 cells redox status, leading to oxidant species production and a decrease of GSH levels, after 1 h exposure, followed by an increase of cytokines production, IL-6 and IL-8, after 24 h. Oxysterol cell treatment also induced after 48 h an increase of NO release, due to the induction of iNOS. Pretreatment with the phenolic extract counteracted oxysterols effects, at least in part by modulating one of the main pathways activated in the cellular response to the action of oxysterols, the MAPK-NF-kB pathway. We demonstrated the ability of the phenolic extract to directly modulate p38 and JNK1/2 phosphorylation and activation of NF-kB, following its inhibitor IkB phosphorylation. The phenolic extract also inhibited iNOS induction, keeping NO concentration at the control level. Our results suggest a protective effect at intestinal level of extra virgin olive oil polyphenols, able to prevent or limit redox unbalance and the onset and progression of chronic intestinal inflammation.

Free Radical Biology and Medicine

Atherosclerosis is currently understood to be mainly the consequence of a complicated inflammator... more Atherosclerosis is currently understood to be mainly the consequence of a complicated inflammatory process at the different stages of plaque development. Among the several inflammatory molecules involved, up-regulation of the functional cyclooxygenase 2/membrane-bound prostaglandin E synthase 1 (COX-2/mPGES-1) axis plays a key role in plaque development. Excessive production of oxidized lipids, following low-density lipoprotein (LDL) oxidation, is a characteristic feature of atherosclerosis. Among LDLs, the oxysterol 27-hydroxycholesterol (27-OH) and the aldehyde 4-hydroxynonenal (HNE) substantially accumulate in the atherosclerotic plaque, contributing to its progression and instability through a variety of processes. This study shows that 27-OH and HNE promote up-regulation of both the inducible enzymes COX-2 and mPGES-1, leading to increased production of prostaglandin (PG) E2 and inducible nitric oxide synthase, and the subsequent release of nitric oxide in human promonocytic U937 cells. The study also examined the potential involvement of the functionally coupled COX-2/mPGES-1 in enhancing the production of certain pro-inflammatory cytokines and of matrix metalloproteinase 9 by U937 cells. This enhancement is presumably due to the induction of PGE2 synthesis, as a result of the up-regulation of the COX-2/mPGES-1, stimulated by the two oxidized lipids, 27-OH and HNE. Induction of PGE2 synthesis might thus be a mechanism of plaque instability and eventual rupture, contributing to matrix metalloproteinase production by activated macrophages.

Free Radical Biology and Medicine

Colorectal cancer (CRC) is one of the most common tumors world wild. High cholesterol diet is con... more Colorectal cancer (CRC) is one of the most common tumors world wild. High cholesterol diet is considered a risk for CRC development. Cholesterol oxidation products, namely oxysterols, have been shown to have a role in human degenerative diseases, mainly for their ability to favor inflammatory reactions. Therefore, they could be involved in inducing intestinal inflammation, a process strongly associated to colorectal carcinogenesis. Dietary oxysterols could derange intestinal epithelial barrier by inducing the activation of metalloproteinases (MMPs) as well as the decrease of tight junctions (TJs), which are essential in mucosa barrier maintenance. Enterocyte-like CaCo-2 cells were treated with a mixture of oxysterols representative of a hyper-cholesterol diet. The time course study of MMPs activity (MMP-9 and MMP-2) showed their significant increase, reaching the maximum at 72 hours treatment. Moreover, the dietary oxysterols decreased the TJ production, in particular of junctional adhesion molecule (JAM), zonula occludens (ZO) and occludin. These two events appeared to be associated. In fact, cell pretreatment with specific MMPs inhibitors restored TJ levels. Therefore, dietary oxysterols could be actually implicated in tumor progression towards a more aggressive phenotype by inducing extracellular matrix destabilization and intestinal barrier disruption.

Free Radical Biology and Medicine

A still growing bulk of evidence underlies the multifaceted biochemical properties of oxysterols,... more A still growing bulk of evidence underlies the multifaceted biochemical properties of oxysterols, several of them of clear relevance to human pathophysiology. Taken up by cells through both vesicular and non vesicular ways or often generated intracellularly, oxysterols contribute to modulate the inflammatory response of a given tissue but also cell viability, metabolism and function. The signaling pathways and the transcription factors whose activation they can influence, often through redox-mediated reactions, are quite a number. Moreover, the outcome of the complex network of intracellular reactions promoted by oxysterols appears to be largely dependent upon specific features and dynamic conditions of the cellular and tissue environment but also on their actual concentration in the site of action. Here focus was given to their potential implication in the advanced progression of atherosclerosis, whosehypercholesterolemia is a recognized primary risk factor. A significant contribution to the possible rupture of atherosclerotic plaque could be provided by defined oxysterols, in particular 27-hydroxycholesterol. Indeed, the latter oxysterol was shown in a promonocytic cell line to amplify the inflammatory reaction and activate several pathways leading to a marked up-regulation of metalloproteases, the key enzymes involved in cap matrix degradation and weakening.

Current medicinal chemistry, Jan 19, 2017

A defective mucosal barrier function is the principal cause of the uncontrolled onset and progres... more A defective mucosal barrier function is the principal cause of the uncontrolled onset and progression of a number of human inflammatory gut diseases, most of which are characterized by chronic intermittent immune and inflammatory responses leading to structural intestinal damage, which can represent a potential risk for colorectal cancer development. During the active disease phase the production of pro-inflammatory cytokines and chemokines, and the induction of oxidative reactions by activated leukocytes and epithelial cells represent the main event in intestinal inflammation. Oxidative stress plays a key role in the development of intestinal damage. Indeed reactive oxygen species and their oxidized by-products regulate redox-sensitive signaling pathways and transcription factors, which sustain inflammation within the intestinal layer. Polyunsaturated fatty acids and cholesterol are the principal targets of oxidative modifications. These lipids, which are cell membrane constituents...

Redox biology, Aug 23, 2017

Oxysterols are known pleiotropic molecules whose antiviral action has been recently discovered. H... more Oxysterols are known pleiotropic molecules whose antiviral action has been recently discovered. Here reported is the activity of a panel of oxysterols against HSV-1 with the identification of a new mechanism of action. A marked antiviral activity not only of 25HC but also of 27HC against HSV-1 was observed either if the oxysterols were added before or after infection, suggesting an activity unrelated to the viral entry inhibition as proposed by previous literature. Therefore, the relation between the pro-inflammatory activity of oxysterols and the activation of NF-kB and IL-6 induced by HSV-1 in the host cell was investigated. Indeed, cell pre-incubation with oxysterols further potentiated IL-6 production as induced by HSV-1 infection with a consequent boost of the interleukin's total cell secretion. Further, a direct antiviral effect of IL-6 administration to HSV-1 infected cells was demonstrated, disclosing an additional mechanism of antiviral action by both 25HC and 27HC.

Free Radical Biology and Medicine, 2017

Consistent experimental data suggest the importance of inflammation-associated oxidative stress i... more Consistent experimental data suggest the importance of inflammation-associated oxidative stress in colorectal cancer (CRC) pathogenesis. Inflammatory bowel disease with chronic intestinal inflammation is now considered a precancerous condition. Oxidative stress is an essential feature of inflammation. Activation of redox-sensitive pro-inflammatory cell signals and inflammatory mediators concur to establish a protumoral environment. In this frame, lipid oxidation products, namely 4-hydroxynonenal and oxysterols, can be produced in big quantity so as to be able to exert their function as inducers of cell signaling pathways of proliferation and survival. Notably, an important source of these two compounds is represented by a high fat diet, which is undoubtedly a risk factor for inflammation and CRC development. Current evidence for the emerging implication of these two oxidized lipids in inflammation and CRC development is discussed in this review.