Hydroxytyrosol Counteracts Triple Negative Breast Cancer Cell Dissemination via Its Copper Complexing Properties.

Polyphenols have gained increasing attention for their therapeutic potential, particularly in conditions like cancer, due to their established antioxidant and anti-inflammatory properties. Recent research highlights their ability to bind to transition metals, such as copper. This is particularly noteworthy given the key role of copper both in the initiation and progression of cancer. Copper can modulate the activity of kinases required for the epithelial-mesenchymal transition (EMT), a process fundamental to tumor cell dissemination. We have previously demonstrated the copper-binding capacity of oleuropein, a secoiridoid found in Olea europaea. In the present study, we investigated the effect of hydroxytyrosol, the primary oleuropein metabolite, on the metastatic potential of three triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and SUM159). We found that hydroxytyrosol modulated the intracellular copper levels, influencing both the epithelial and mesenchymal markers, by downregulating copper-dependent AKT phosphorylation, a member of the EMT signaling cascade, through Western blot, RT-qPCR, and immunofluorescence. Indeed, by optical spectra, EPR, and in silico approaches, we found that hydroxytyrosol formed a complex with copper, acting as a chelating agent, thus regulating its homeostasis and affecting the copper-dependent signaling cascades. While our results bring to light the copper-chelating properties of hydroxytyrosol capable of countering tumor progression, they also provide further confirmation of the key role of copper in promoting the aggressiveness of triple-negative breast cancer cells.

AKT-driven epithelial-mesenchymal transition is affected by copper bioavailability in HER2 negative breast cancer cells via a LOXL2-independent mechanism.

BACKGROUND: The main mechanism underlying cancer dissemination is the epithelial to mesenchymal transition (EMT). This process is orchestrated by cytokines like TGFß, involving "non-canonical" AKT- or STAT3-driven pathways. Recently, the alteration of copper homeostasis seems involved in the onset and progression of cancer. METHODS: We expose different breast cancer cell lines, including two triple negative (TNBC) ones, an HER2 enriched and one cell line representative of the Luminal A molecular subtype, to short- or long-term copper-chelation by triethylenetetramine (TRIEN). We analyse changes in the expression of EMT markers (E-cadherin, fibronectin, vimentin and αSMA), in the levels and activity of extracellular matrix components (LOXL2, fibronectin and MMP2/9) and of copper homeostasis markers by Western blot analyses, immunofluorescence, enzyme activity assays and RT-qPCR. Boyden Chamber and wound healing assays revealed the impact of copper chelation on cell migration. Additionally, we explored whether perturbation of copper homeostasis affects EMT prompted by TGFß. Metabolomic and lipidomic analyses were applied to search the effects of copper chelation on the metabolism of breast cancer cells. Finally, bioinformatics analysis of data on breast cancer patients obtained from different databases was employed to correlate changes in kinases and copper markers with patients' survival. RESULTS: Remarkably, only HER2 negative breast cancer cells differently responded to short- or long-term exposure to TRIEN, initially becoming more aggressive but, upon prolonged exposure, retrieving epithelial features, reducing their invasiveness. This phenomenon may be related to the different impact of the short and prolonged activation of the AKT kinase and to the repression of STAT3 signalling. Bioinformatics analyses confirmed the positive correlation of breast cancer patients' survival with AKT activation and up-regulation of CCS. Eventually, metabolomics studies demonstrate a prevalence of glycolysis over mitochondrial energetic metabolism and of lipidome changes in TNBC cells upon TRIEN treatment. CONCLUSIONS: We provide evidence of a pivotal role of copper in AKT-driven EMT activation, acting independently of HER2 in TNBC cells and via a profound change in their metabolism. Our results support the use of copper-chelators as an adjuvant therapeutic strategy for TNBC.

Ficus carica L photodermatitis: a report of five cases with histopathologic study and review of the literature.

Phytophotodermatitis is a condition caused by contamination of the skin with phototoxic plant substances, followed by exposure to ultraviolet rays. Ficus carica L 1753, belonging to the Moraceae family, can be responsible for acute photodermatitis. We present five cases of photodermatitis caused by contact with Ficus carica L and subsequent exposure to sunlight. A histopathologic study and review of the literature are included.

Copper-Dependent Kinases and Their Role in Cancer Inception, Progression and Metastasis.

In recent years, copper function has been expanded beyond its consolidated role as a cofactor of enzyme catalysis. Recent papers have demonstrated a new dynamic role for copper in the regulation of cell signaling pathways through direct interaction with protein kinases, modulating their activity. The activation of these pathways is exacerbated in cancer cells to sustain the different steps of tumor growth and dissemination. This review will focus on a novel proposed role for the transition metal copper as a regulator of cell signaling pathways through direct interaction with known protein kinases, which exhibit binding domains for this metal. Activation of these pathways in cancer cells supports both tumor growth and dissemination. In addition to the description of the results recently reported in the literature on the subject, relevance will be given to the possibility of controlling the cellular levels of copper and its homeostatic regulators. Overall, these findings may be of central relevance in order to propose copper and its homeostatic regulators as possible targets for novel therapies, which may act synergistically to those already existing to control cancer growth and dissemination.

Copper in tumors and the use of copper-based compounds in cancer treatment.

Copper homeostasis is strictly regulated by protein transporters and chaperones, to allow its correct distribution and avoid uncontrolled redox reactions. Several studies address copper as involved in cancer development and spreading (epithelial to mesenchymal transition, angiogenesis). However, being endogenous and displaying a tremendous potential to generate free radicals, copper is a perfect candidate, once opportunely complexed, to be used as a drug in cancer therapy with low adverse effects. Copper ions can be modulated by the organic counterpart, after complexed to their metalcore, either in redox potential or geometry and consequently reactivity. During the last four decades, many copper complexes were studied regarding their reactivity toward cancer cells, and many of them could be a drug choice for phase II and III in cancer therapy. Also, there is promising evidence of using 64Cu in nanoparticles as radiopharmaceuticals for both positron emission tomography (PET) imaging and treatment of hypoxic tumors. However, few compounds have gone beyond testing in animal models, and none of them got the status of a drug for cancer chemotherapy. The main challenge is their solubility in physiological buffers and their different and non-predictable mechanism of action. Moreover, it is difficult to rationalize a structure-based activity for drug design and delivery. In this review, we describe the role of copper in cancer, the effects of copper-complexes on tumor cell death mechanisms, and point to the new copper complexes applicable as drugs, suggesting that they may represent at least one component of a multi-action combination in cancer therapy.

Iron Serum Markers Profile in Frontotemporal Lobar Degeneration.

Frontotemporal lobar degeneration (FTLD) is a progressive neurodegenerative syndrome. Defects of copper (Cu) and iron (Fe) homeostasis are involved in the development of several neurodegenerative diseases and their homeostasis is interconnected by the Cu-protein ceruloplasmin (Cp), responsible for Fe oxidative state. In this study we assessed Fe, transferrin (Trf), ferritin, Cp specific activity (eCp/iCp), Cp/Trf ratio, and Trf saturation in 60 FTLD patients and 43 healthy controls, and discussed the results in relation to Cu homeostasis. The significant decrease of the eCp/iCp in the FTLD patients supports the involvement of Fe imbalance in the onset and progression of FTLD.

Polymorphic Genetic Markers of the GABA Catabolism Pathway in Alzheimer's Disease.

BACKGROUND: The compilation of a list of genetic modifiers in Alzheimer's disease (AD) is an open research field. The GABAergic system is affected in several neurological disorders but its role in AD is largely understudied. OBJECTIVE/METHODS: As an explorative study, we considered variants in genes of GABA catabolism (ABAT, ALDH5A1, AKR7A2), and APOE in 300 Italian patients and 299 controls. We introduce a recent multivariate method to take into account the individual APOE genotype, thus controlling for the effect of the discrepant allele distributions in cases versus controls. We add a genotype-phenotype analysis based on age at onset and the Mini-Mental State Evaluation score. RESULTS: On the background of strongly divergent APOE allele distributions in AD versus controls, two genotypic interactions that represented a subtle but significant peculiarity of the AD cohort emerged. The first is between ABAT and APOE, and the second between some ALDH5A1 genotypes and APOE. Decreased SSADH activity is predicted in AD carriers of APOEɛ4, representing an additional suggestion for increased oxidative damage. CONCLUSION: We identified a difference between AD and controls, not in a shift of the allele frequencies at genes of the GABA catabolism pathway, but rather in gene interactions peculiar of the AD cohort. The emerging view is that of a multifactorial contribution to the disease, with a main risk factor (APOE), and additional contributions by the variants here considered. We consider genes of the GABA degradation pathway good candidates as modifiers of AD, contributing to energy impairment in AD brain.

SSADH Variants Increase Susceptibility of U87 Cells to Mitochondrial Pro-Oxidant Insult.

Succinate semialdehyde dehydrogenase (SSADH) is a mitochondrial enzyme, encoded by ALDH5A1, mainly involved in γ-aminobutyric acid (GABA) catabolism and energy supply of neuronal cells, possibly contributing to antioxidant defense. This study aimed to further investigate the antioxidant role of SSADH, and to verify if common SNPs of ALDH5A1 may affect SSADH activity, stability, and mitochondrial function. In this study, we used U87 glioblastoma cells as they represent a glial cell line. These cells were transiently transfected with a cDNA construct simultaneously harboring three SNPs encoding for a triple mutant (TM) SSADH protein (p.G36R/p.H180Y/p.P182L) or with wild type (WT) cDNA. SSADH activity and protein level were measured. Cell viability, lipid peroxidation, mitochondrial morphology, membrane potential (ΔΨ), and protein markers of mitochondrial stress were evaluated upon Paraquat treatment, in TM and WT transfected cells. TM transfected cells show lower SSADH protein content and activity, fragmented mitochondria, higher levels of peroxidized lipids, and altered ΔΨ than WT transfected cells. Upon Paraquat treatment, TM cells show higher cell death, lipid peroxidation, 4-HNE protein adducts, and lower ΔΨ, than WT transfected cells. These results reinforce the hypothesis that SSADH contributes to cellular antioxidant defense; furthermore, common SNPs may produce unstable, less active SSADH, which could per se negatively affect mitochondrial function and, under oxidative stress conditions, fail to protect mitochondria.

Endolimax nana and urticaria.

Endolimax nana is a commensal protozoan of the colon. We report a case of chronic urticaria associated with E. nana in a 34-year-old Italian woman. The patient suffered from abdominal pain, diarrhoea and weight loss. The disease appeared after a trip to Vietnam. Laboratory examinations showed mild blood eosinophilia. Three coproparasitological examinations were positive for cysts of E. nana. The patient was successfully treated with two courses of metronidazole (2 g/day for 10 days each). No antihistamines were used. Three coproparasitological examinations, carried out at the end of the therapy, were negative. Follow up (six months) was negative. E. nana can be responsible for very rare cases of abdominal pain, diarrhoea, polyarthritis and urticaria.

Radiotherapy of primary cutaneous anaplastic large cell lymphoma: our experience in 30 cases.

BACKGROUND: Since the observation of new cases of primary cutaneous anaplastic large cell lymphoma (PCALCL) with a seemingly aggressive clinical presentation and a favorable response to radiation therapy (RT), a review of our series has been performed to evaluate the results of RT. MATERIALS AND METHODS: The study is a retrospective analysis of 30 patients with PCALCL treated with conventional energy RT. RESULTS: About 55 fields of irradiation were performed. Complete clinical response (CCR) was obtained in 29 cases and a partial clinical response (PCR) in one. Two lesions had a marginal relapse at 7 and 37 months, respectively. In eight cases, new lesions appeared in the same body district and in six cases, in body sites at distance from the field of treatment. Total radiation dose ranged from 15 to 35 Gy (median 25 Gy). Follow-up range was 2-218 months (median 38.5 months) with a local control rate of 85% and a relapse-free rate of 49% at 5 years from RT. Three cases developed a nodal involvement. CONCLUSIONS: PCALCL belongs to the group of primary cutaneous CD30+ lymphoproliferative disorders, accounting for 25-30% of cutaneous T-cell lymphomas. RT is considered of choice as alternative to surgical excision of solitary lesions. Recent studies have focused on ideal dose to obtain local control indicating 30 Gy as adequate, but others have hypothesized that lower doses may suffice. Our study confirms the excellent role of RT in the local control of the disease with total doses ≤25 Gy.

Paclitaxel as first- or second-line treatment for HIV-negative Kaposi's sarcoma: a retrospective study of 58 patients.

Background: Paclitaxel has recently been approved for AIDS-related Kaposi's sarcoma (KS) and there is much interest also in HIV-negative KS.Objective: To assess the safety and effectiveness of intravenous paclitaxel in the treatment of non-HIV-associated KS.Method: A retrospective database analysis of our departmental database in histologically proven, HIV-negative KS.Results: Fifty-eight patients treated with intravenous paclitaxel 100 mg weekly were identified. Among these patients, 11 patients underwent paclitaxel as first-line treatment, whereas 47 received paclitaxel after other types of systemic chemotherapy. Fifty-three (94.6%) patients achieved a partial or a complete remission after a mean of 13.5 infusions. Disease progression was observed in two patients and one patient had a stable disease. Thirty-one (58.5%) of 53 responding patients are still stable after a mean of 19.1 months of follow-up, while 22 (41.5%) patients relapsed after a mean of 14 months. Paclitaxel was repeated in relapsed patients obtaining PR/CR in all cases. Tolerance was good except for one patient who discontinued the treatment because of a severe allergic reaction.Conclusion: Paclitaxel is effective for the treatment of non-HIV-related KS, both as first- and as second-line treatment. It is well tolerated and can be repeated without loss of efficacy.

Copper homeostasis as target of both consolidated and innovative strategies of anti-tumor therapy.

BACKGROUND: Copper was reported to be involved in the onset and progression of cancer. Proteins in charge of copper uptake and distribution, as well as cuproenzymes, are altered in cancer. More recently, proteins involved in signaling cascades, regulating cell proliferation, and anti-apoptotic protein factors were found to interact with copper. Therefore, therapeutic strategies using copper complexing molecules have been proposed for cancer therapy and used in clinical trials. OBJECTIVES: This review will focus on novel findings about the involvement of copper and cupro-proteins in cancer dissemination process, epithelium to mesenchymal transition and vascularization. Particularly, implication of well-established (e.g. lysil oxidase) or newly identified copper-binding proteins (e.g. MEMO1), as well as their interplay, will be discussed. Moreover, we will describe recently synthesized copper complexes, including plant-derived ones, and their efficacy in contrasting cancer development. CONCLUSIONS: The research on the involvement of copper in cancer is still an open field. Further investigation is required to unveil the mechanisms involved in copper delivery to the novel copper-binding proteins, which may identify other possible gene and protein targets for cancer therapy.

Succinic semialdehyde dehydrogenase deficiency: The combination of a novel ALDH5A1 gene mutation and a missense SNP strongly affects SSADH enzyme activity and stability.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare autosomal recessive metabolic disorder of GABA catabolism. SSADH is a mitochondrial homotetrameric enzyme encoded by ALDH5A1 gene. We report the molecular characterization of ALDH5A1 gene in an Italian SSADHD patient, showing heterozygosity for four missense mutations: c.526G>A (p.G176R), c.538C>T (p.H180Y), c.709G>T (p.A237S) and c.1267A>T (p.T423S), the latter never described so far. The patient inherited c.526A in cis with c.538T from the mother and c.709T in cis with c.1267T from the father. To explore the effects of the two allelic arrangements on SSADH activity and protein level, wild type, single or double mutated cDNA constructs were expressed in a cell system. The p.G176R change, alone or in combination with p.H180Y, causes the abolishment of enzyme activity. Western blot analysis showed a strongly reduced amount of the p.176R-p.180Y double mutant protein, suggesting increased degradation. Indeed, in silico analyses confirmed high instability of this mutant homotetramer. Enzyme activity relative to the other p.423S-p.237S double mutant is around 30% of wt. Further in silico analyses on all the possible combinations of mutant monomers suggest the lowest stability for the tetramer constituted by p.176R-p.180Y monomers and the highest stability for that constituted by p.237S-p.423S monomers. The present study shows that when a common SNP, associated with a slight reduction of SSADH activity, is inherited in cis with a mutation showing no consequences on the enzyme function, the activity is strongly affected. In conclusion, the peculiar arrangement of four missense mutations occurring in this patient is responsible for the SSADHD phenotype.

Oleuropein shows copper complexing properties and noxious effect on cultured SH-SY5Y neuroblastoma cells depending on cell copper content.

The secoiridoid oleuropein is a non-flavonoid polyphenol, found in the fruit, leaves and food derivatives from Olea europea. Like other polyphenols it shows a very low toxicity towards healthy tissues and a protective action against cancer or neurodegeneration, but its mechanism of action is not yet understood. In the present report we have used optical and ESR spectroscopy as well as molecular modelling to demonstrate that oleuropein forms a complex with the transition metal copper; the dysmetabolism of this metal is suspected to be involved in both cancer and neurodegeneration. Experiments carried out with the aglycon derivative of oleuropein, produced by ß-glycosidase treatment of oleuropein glycoside, showed that also the aglycon forms copper-complexes, but with different spectroscopic features than the glycosidic form. Molecular modelling analysis confirmed that two oleuropein molecules (glycosidic or aglycon forms) can easily coordinate one copper ion. The relationship between oleuropein and copper was investigated in SH-SY5Y human neuroblastoma cells. When cells were depleted of copper by treatment with the copper chelator triethylenetetramine (Trien), that binds copper with higher affinity than oleuropein, oleuropein was less toxic than to copper-adequate cells. Conversely, incubation of SH-SY5Y cells with exogenous copper sulphate increased cell susceptibility to oleuropein. Furthermore SH-SY5Y cells differentiated by retinoic acid pre-treatment showed a lower level of copper, and were more resistant to oleuropein treatment. The oleuropein aglycon was not toxic towards SH-SY5Y cells. In conclusion, the copper-oleuropein complex may be involved in the toxicity of oleuropein towards tumour cells, depending on their copper level.

SSADH deficiency in an Italian family: a novel ALDH5A1 gene mutation affecting the succinic semialdehyde substrate binding site.

SSADH deficiency (SSADHD) is a rare autosomal recessively inherited metabolic disorder. It is associated with mutations of ALDH5A1 gene, coding for the homotetrameric enzyme SSADH. This enzyme is involved in γ-aminobutyric acid (GABA) catabolism, since it oxidizes succinic semialdehyde (SSA) to succinate. Mutations in ALDH5A1 gene result in the abnormal accumulation of γ-hydroxybutyrate (GHB), which is pathognomonic of SSADHD. In the present report, diagnosis of SSADHD in a three-month-old female was achieved by detection of high levels of GHB in urine. Sequence analysis of ALDH5A1 gene showed that the patient was a compound heterozygote for c.1226G > A (p.G409D) and the novel missense mutation, c.1498G > C (p.V500 L). By ALDH5A1 gene expression in transiently transfected HEK293 cells and enzyme activity assays, we demonstrate that the p.V500 L mutation, despite being conservative, produces complete loss of enzyme activity. In silico protein modelling analysis and evaluation of tetramer destabilizing energies suggest that structural impairment and partial occlusion of the access channel to the active site affect enzyme activity. These findings add further knowledge on the missense mutations associated with SSADHD and the molecular mechanisms underlying the loss of the enzyme activity.

Are topical retinoids teratogenic?

Retinoic acid is a physiological compound of human blood. Blood levels range from 1000 to 7000 pg/mL (usually 1500-5000 pg/mL). Results of studies on absorption of topical retinoic acid in laboratory animals, although rather conflicting, demonstrate that it induces plasma concentrations which are well below concentrations caused by non-teratogenic oral doses. In humans, minimal percutaneous absorption of tretinoin was observed after topical applications. Neither single dose nor long-term treatment with topical tretinoin affect the endogenous levels of retinoic acid or its metabolites. Topical application of tretinoin at doses used in acne unlikely induces systemic effects. Although some clinical cases of suspected tretinoin-related embryotoxicity have been described, three prospective cohort studies clearly demonstrated the safety of topical tretinoin as an embryotoxic agent.

A Second Case of Gobello Nevus Syndrome.

An uncommon type of epidermal nevus characterized by hyperpigmented hyperkeratotic bands following a Blaschko-linear pattern and generalized follicular hyperkeratosis were observed in a 17-year-old male patient who additionally showed tufted hair folliculitis on the scalp and clinodactyly of the fifth finger of both hands. The combination of epidermal nevus with skeletal abnormalities was first described by Gobello et al. [Dermatology 2000;201:51-55] as a new epidermal nevus syndrome that was named after the first author of this work. Our case shows identical clinical and histopathological features and represents the second case of this rare syndrome reported in the literature.