Paola De Cicco

Butyrate Attenuates Lipopolysaccharide-Induced Inflammation in Intestinal Cells and Crohn's Mucosa through Modulation of Antioxidant Defense Machinery

Oxidative stress plays an important role in the pathogenesis of inflammatory bowel disease (IBD), including Crohns disease (CrD). High levels of Reactive Oxygen Species (ROS) induce the activation of the redox-sensitive nuclear transcription factor kappa-B (NF-κB), which in turn triggers the inflammatory mediators. Butyrate decreases pro-inflammatory cytokine expression by the lamina propria mononuclear cells in CrD patients via inhibition of NF-κB activation, but how it reduces inflammation is still unclear. We suggest that butyrate controls ROS mediated NF-κB activation and thus mucosal inflammation in intestinal epithelial cells and in CrD colonic mucosa by triggering intracellular antioxidant defense systems. Intestinal epithelial Caco-2 cells and colonic mucosa from 14 patients with CrD and 12 controls were challenged with or without lipopolysaccaride from Escherichia Coli (EC-LPS) in presence or absence of butyrate for 4 and 24 h. The effects of butyrate on oxidative stress, p42/44 MAP kinase phosphorylation, p65-NF-κB activation and mucosal inflammation were investigated by real time PCR, western blot and confocal microscopy. Our results suggest that EC-LPS challenge induces a decrease in Gluthation-S-Transferase-alpha (GSTA1/A2) mRNA levels, protein expression and catalytic activity; enhanced levels of ROS induced by EC-LPS challenge mediates p65-NF-κB activation and inflammatory response in Caco-2 cells and in CrD colonic mucosa. Furthermore butyrate treatment was seen to restore GSTA1/A2 mRNA levels, protein expression and catalytic activity and to control NF-κB activation, COX-2, ICAM-1 and the release of pro-inflammatory cytokine. In conclusion, butyrate rescues the redox machinery and controls the intracellular ROS balance thus switching off EC-LPS induced inflammatory response in intestinal epithelial cells and in CrD colonic mucosa.

Role of the cystathionine γ lyase/hydrogen sulfide pathway in human melanoma progression.

In humans, two main metabolic enzymes synthesize hydrogen sulfide (H2S): cystathionine γ lyase (CSE) and cystathionine β synthase (CBS). A third enzyme, 3‐mercaptopyruvate sulfurtransferase (3‐MST), synthesizes H2S in the presence of the substrate 3‐mercaptopyruvate (3‐MP). The immunohistochemistry analysis performed on human melanoma samples demonstrated that CSE expression was highest in primary tumors, decreased in the metastatic lesions and was almost silent in non‐lymph node metastases. The primary role played by CSE was confirmed by the finding that the overexpression of CSE induced spontaneous apoptosis of human melanoma cells. The same effect was achieved using different H2S donors, the most active of which was diallyl trisulfide (DATS). The main pro‐apoptotic mechanisms involved were suppression of nuclear factor‐κB activity and inhibition of AKT and extracellular signal‐regulated kinase pathways. A proof of concept was obtained in vivo using a murine melanoma model. In fact, either l‐cysteine, the CSE substrate, or DATS inhibited tumor growth in mice. In conclusion, we have determined that the l‐cysteine/CSE/H2S pathway is involved in melanoma progression.

ATB-346, a novel hydrogen sulfide-releasing anti-inflammatory drug, induces apoptosis of human melanoma cells and inhibits melanoma development in vivo

Inflammation plays a key role in tumor promotion and development. Indeed, cyclooxygenase-2 (COX-2) expression is strongly associated with different types of cancer. An emerging class of compounds with significant anti-inflammatory properties is the hydrogen sulfide-releasing non-steroidal anti-inflammatory drugs (H2S-NSAIDs). They consist of a traditional NSAID to which an H2S-releasing moiety is covalently attached. We have recently demonstrated that H2S donors inhibit melanoma cell proliferation. In the current study, we evaluated the potential beneficial effects of a new H2S-releasing derivative of naproxen, ATB-346 [2-(6-methoxynapthalen-2-yl)-propionic acid 4-thiocarbamoyl phenyl ester] which inhibits COX activity but also releases H2S. We used cell culture and a mouse melanoma model to evaluate the effect of ATB-346 on: i) in vitro growth of human melanoma cells; ii) in vivo melanoma development in mice. Cell culture studies demonstrated that ATB-346 reduced the in vitro proliferation of human melanoma cells and this effect was associated to induction of apoptosis and inhibition of NF-κB activation. Moreover, ATB-346 had novel Akt signaling inhibitory properties. Daily oral dosing of ATB-346 (43μmol/kg) significantly reduced melanoma development in vivo. This study shows that ATB-346, a novel H2S-NSAID, inhibits human melanoma cell proliferation by inhibiting pro-survival pathways associated with NF-κB and Akt activation. Furthermore, oral treatment with ATB-346 inhibits melanoma growth in mice. In conclusion, the combination of inhibition of cyclooxygenase and delivery of H2S by ATB-346 may offer a promising alternative to existing therapies for melanoma.

Differential expression of cyclooxygenase-2 in metastatic melanoma affects progression free survival

The possible correlation between cyclooxygenase-2 (COX-2) expression and disease progression in melanoma is still a matter of debate. Analysis of COX-2 expression in 45 lymph node melanoma metastases demonstrates a significant correlation between the percent of expression and progression free survival (PFS). A positive COX-2 expression ≥10% (COX-2high), as opposite to a positive expression ≤9% (COX-2low), translated into a striking significant reduction of PFS of about 3 years. The reduction in PFS correlated neither with BRAFV600E nor with NRASQ61 expression in the analyzed samples. This concept was reinforced by the finding that tumour development in COX-2−/− mice was almost blunted. Similarly, inhibition of COX-2 protein expression in human melanoma cell lines, by using siRNAs technology as well as selective inhibition of COX-2 activity by celecoxib, reduced cellular proliferation and invasiveness. In conclusion we show that COX-2high is a negative prognostic factor in metastatic melanoma. Our study also clarifies that the uncertainty about the role of COX-2 in metastatic malignant melanoma, found in the current relevant literature, is probably due to the fact that a threshold in COX-2 expression has to be reached in order to impact on cancer malignancy. Our findings suggest that COX-2 expression may become an useful diagnostic tool in defining melanoma malignancy as well as argue for a possible therapeutic use of NSAID as add on therapy in selected cases.

The Hydrogen Sulfide Releasing Molecule Acetyl Deacylasadisulfide Inhibits Metastatic Melanoma

Melanoma is the most common form of skin cancer. Given its high mortality, the interest in the search of preventive measures, such as dietary factors, is growing significantly. In this study we tested, in vitro and in vivo, the potential anti-cancer effect of the acetyl deacylasadisulfide (ADA), a vinyl disulfide compound, isolated and purified from asafoetida a foul-smelling oleo gum-resin of dietary and medicinal relevance. ADA markedly suppressed proliferation of human melanoma cell lines by inducing apoptosis. Moreover, treatment of melanoma cells with ADA reduced nuclear translocation and activation of NF-κB, decreased the expression of the anti-apoptotic proteins c-FLIP, XIAP, and Bcl-2 and inhibited the phosphorylation and activation of both AKT and ERK proteins, two of the most frequently deregulated pathways in melanoma. Finally, the results obtained in vitro were substantiated by the findings that ADA significantly and dose-dependently reduced lung metastatic foci formation in C57BL/6 mice. In conclusion, our findings suggest that ADA significantly inhibits melanoma progression in vivo and could represent an important lead compound for the development of new anti-metastatic agents.

NMR-based phytochemical analysis of Vitis vinifera cv Falanghina leaves. Characterization of a previously undescribed biflavonoid with antiproliferative activity

Vitis vinifera cv Falanghina is an ancient grape variety of Southern Italy. A thorough phytochemical analysis of the Falanghina leaves was conducted to investigate its specialised metabolite content. Along with already known molecules, such as caftaric acid, quercetin-3-O-β-d-glucopyranoside, quercetin-3-O-β-d-glucuronide, kaempferol-3-O-β-d-glucopyranoside and kaempferol-3-O-β-d-glucuronide, a previously undescribed biflavonoid was identified. For this last compound, a moderate bioactivity against metastatic melanoma cells proliferation was discovered. This datum can be of some interest to researchers studying human melanoma. The high content in antioxidant glycosylated flavonoids supports the exploitation of grape vine leaves as an inexpensive source of natural products for the food industry and for both pharmaceutical and nutraceutical companies. Additionally, this study offers important insights into the plant physiology, thus prompting possible technological researches of genetic selection based on the vine adaptation to specific pedo-climatic environments.

Indicaxanthin from Opuntia Ficus Indica (L. Mill) impairs melanoma cell proliferation, invasiveness, and tumor progression

BACKGROUND A strong, reciprocal crosstalk between inflammation and melanoma has rigorously been demonstrated in recent years, showing how crucial is a pro-inflammatory microenvironment to drive therapy resistance and metastasis. PURPOSE We investigated on the effects of Indicaxanthin, a novel, anti-inflammatory and bioavailable phytochemical from Opuntia Ficus Indica fruits, against human melanoma both in vitro and in vivo. STUDY DESIGN AND METHODS The effects of indicaxanthin were evaluated against the proliferation of A375 human melanoma cell line and in a mice model of cutaneous melanoma. Cell proliferation was assessed by MTT assay, apoptosis by Annexin V-Fluorescein Isothiocyanate/Propidium Iodide staining, protein expression by western blotting, melanoma lesions were subcutaneously injected in mice with B16/F10 cells, chemokine release was quantified by ELISA. RESULTS Data herein presented demonstrate that indicaxanthin effectively inhibits the proliferation of the highly metastatic and invasive A375 cells as shown by growth inhibition, apoptosis induction and cell invasiveness reduction. More interestingly, in vitro data were paralleled by those in vivo showing that indicaxanthin significantly reduced tumor development when orally administered to mice. The results of our study also clarify the molecular mechanisms underlying the antiproliferative effect of indicaxanthin, individuating the inhibition of NF-κB pathway as predominant. CONCLUSION In conclusion, we demonstrated that indicaxanthin represents a novel phytochemical able to significantly inhibit human melanoma cell proliferation in vitro and to impair tumor progression in vivo. When considering the resistance of melanoma to the current therapeutical approach and the very limited number of phytochemicals able to partially counteract it, our findings may be of interest to explore indicaxanthin potential in further and more complex melanoma studies in combo therapy, i.e. where different check points of melanoma development are targeted.

Current Practices and Awareness of Anticancer Plants in the Traditional Healthcare System

Nowadays, the increasing burden of cancer has renewed the interest in plants as source of potential anticancer drugs. In fact, one of the major problems with cancer chemotherapy is the extreme toxicity of the traditional drugs. Thus, the research for new biologically active molecules against cancer is targeted on plants and their derived natural products. Plants used in traditional medicine have proved effective and safe in the treatment and management of cancers, and almost 80% of the world’s population use anticancer agents of natural origin. Thus, the aim of the present chapter is to highlight the plant-derived natural products and their analogues established as anticancer agents in the traditional and current medicinal practices, their toxicological effects, and clinical exercises along with the new technologies applied for delivering natural anticancer drugs.

Hydrogen Sulfide Reduces Myeloid-Derived Suppressor Cell-Mediated Inflammatory Response in a Model of Helicobacter hepaticus-Induced Colitis

Chronic inflammation contributes to tumor initiation in colitis-associated colorectal cancer (CRC). Indeed, inflammatory bowel disease (IBD) patients show an increased risk of developing CRC. Cancer immune evasion is a major issue in CRC and preclinical and clinical evidence has defined a critical role for myeloid-derived suppressor cells (MDSCs) that contribute to tumor growth and progression by suppressing T-cells and modulating innate immune responses. MDSCs comprise a heterogeneous population of immature myeloid cells that can be distinct in two subtypes: CD11b+Ly6G+Ly6Clow with granulocytic phenotype (G-MDSCs) and CD11b+Ly6G−Ly6Chigh with monocytic phenotype (M-MDSCs). Hydrogen sulfide (H2S) is an endogenous gaseous signaling molecule that regulates various physiological and pathophysiological functions. In particular, several studies support its anti-inflammatory activity in experimental colitis and ulcer. However, the role of the H2S pathway in innate immune-mediated IBD has not yet been elucidated. To better define a possible link between MDSCs and H2S pathway in colitis-associated CRC development, we used an innate immune-mediated IBD model induced by infection with the bacterium Helicobacter hepaticus (Hh), closely resembling human IBD. Here, we demonstrated an involvement of MDSCs in colitis development. A significant time-dependent increase of both G-MDSCs and M-MDSCs was observed in the colon and in the spleen of Hh-infected mice. Following, we observed that chronic oral administration of the H2S donor DATS reduced colon inflammation by limiting the recruitment of G-MDSCs in the colon of Hh-infected mice. Thus, we identify the metabolic pathway l-cysteine/H2S as a possible new player in the immunosuppressive mechanism responsible for the MDSCs-promoted colitis-associated cancer development.

MicroRNA-143-3p inhibits growth and invasiveness of melanoma cells by targeting cyclooxygenase-2 and inversely correlates with malignant melanoma progression

Graphical abstract Figure. No Caption available. Abstract Malignant melanoma is one of the most leading form of skin cancer associated with a low patient survival rate. Increasing evidence revealed that microRNAs (miRNAs) play a crucial role in the occurrence and development of several form of cancer including melanoma. In this study, we aimed at investigating the expression and role of miR‐143‐3p in human malignant melanoma. Our results showed that the expression of miR‐143‐3p was lower in human melanoma cells, as well as human biopsy specimens, when compared to normal human melanocytes. Ectopic expression of miR‐143‐3p in human melanoma cells inhibited proliferation, migration, invasion and promoted apoptosis acting through a molecular mechanism that, at least in part, is dependent on inhibition of cyclooxygenase‐2 (COX‐2) gene. Collectively, these results demonstrate that miR‐143‐3p could represent at the same time, a new early diagnostic marker and therapeutic target acting as tumor suppressor in melanoma cancer.