Cristiano Farace

Cadmium Modifies the Cell Cycle and Apoptotic Profiles of Human Breast Cancer Cells Treated with 5-Fluorouracil

Industrialisation, the proximity of factories to cities, and human work activities have led to a disproportionate use of substances containing heavy metals, such as cadmium (Cd), which may have deleterious effects on human health. Carcinogenic effects of Cd and its relationship with breast cancer, among other tumours, have been reported. 5-Fluorouracil (5-FU) is a fluoropyrimidine anticancer drug used to treat solid tumours of the colon, breast, stomach, liver, and pancreas. The purpose of this work was to study the effects of Cd on cell cycle, apoptosis, and gene and protein expression in MCF-7 breast cancer cells treated with 5-FU. Cd altered the cell cycle profile, and its effects were greater when used either alone or in combination with 5-FU compared with 5-FU alone. Cd significantly suppressed apoptosis of MCF-7 cells pre-treated with 5-FU. Regarding gene and protein expression, bcl2 expression was mainly upregulated by all treatments involving Cd. The expression of caspase 8 and caspase 9 was decreased by most of the treatments and at all times evaluated. C-myc expression was increased by all treatments involving Cd, especially 5-FU plus Cd at the half time of treatment. Cd plus 5-FU decreased cyclin D1 and increased cyclin A1 expression. In conclusion, our results indicate that exposure to Cd blocks the anticancer effects of 5-FU in MCF-7 cells. These results could have important clinical implications in patients treated with 5-FU-based therapies and who are exposed to high levels of Cd.

Immune cell impact of three differently coated lipid nanocapsules: pluronic, chitosan and polyethylene glycol

Lipid nanocapsules (NCs) represent promising tools in clinical practice for diagnosis and therapy applications. However, the NC appropriate functionalization is essential to guarantee high biocompatibility and molecule loading ability. In any medical application, the immune system-impact of differently functionalized NCs still remains to be fully understood. A comprehensive study on the action exerted on human peripheral blood mononuclear cells (PBMCs) and major immune subpopulations by three different NC coatings: pluronic, chitosan and polyethylene glycol-polylactic acid (PEG) is reported. After a deep particle characterization, the uptake was assessed by flow-cytometry and confocal microscopy, focusing then on apoptosis, necrosis and proliferation impact in T cells and monocytes. Cell functionality by cell diameter variations, different activation marker analysis and cytokine assays were performed. We demonstrated that the NCs impact on the immune cell response is strongly correlated to their coating. Pluronic-NCs were able to induce immunomodulation of innate immunity inducing monocyte activations. Immunomodulation was observed in monocytes and T lymphocytes treated with Chitosan-NCs. Conversely, PEG-NCs were completely inert. These findings are of particular value towards a pre-selection of specific NC coatings depending on biomedical purposes for pre-clinical investigations; i.e. the immune-specific action of particular NC coating can be excellent for immunotherapy applications.

Microenvironmental Modulation of Decorin and Lumican in Temozolomide-Resistant Glioblastoma and Neuroblastoma Cancer Stem-Like Cells

The presence of cancer stem cells (CSCs) or tumor-initiating cells can lead to cancer recurrence in a permissive cell–microenvironment interplay, promoting invasion in glioblastoma (GBM) and neuroblastoma (NB). Extracellular matrix (ECM) small leucine-rich proteoglycans (SLRPs) play multiple roles in tissue homeostasis by remodeling the extracellular matrix (ECM) components and modulating intracellular signaling pathways. Due to their pan-inhibitory properties against receptor tyrosine kinases (RTKs), SLRPs are reported to exert anticancer effects in vitro and in vivo. However, their roles seem to be tissue-specific and they are also involved in cancer cell migration and drug resistance, paving the way to complex different scenarios. The aim of this study was to determine whether the SLRPs decorin (DCN) and lumican (LUM) are recruited in cell plasticity and microenvironmental adaptation of differentiated cancer cells induced towards stem-like phenotype. Floating neurospheres were generated by applying CSC enrichment medium (neural stem cell serum-free medium, NSC SFM) to the established SF-268 and SK-N-SH cancer cell lines, cellular models of GBM and NB, respectively. In both models, the time-dependent synergistic activation of DCN and LUM was observed. The highest DCN and LUM mRNA/protein expression was detected after cell exposure to NSC SFM for 8/12 days, considering these cells as SLRP-expressing (SLRP+) CSC-like. Ultrastructural imaging showed the cellular heterogeneity of both the GBM and NB neurospheres and identified the inner living cells. Parental cell lines of both GBM and NB grew only in soft agar + NSC SFM, whereas the secondary neurospheres (originated from SLRP+ t8 CSC-like) showed lower proliferation rates than primary neurospheres. Interestingly, the SLRP+ CSC-like from the GBM and NB neurospheres were resistant to temozolomide (TMZ) at concentrations >750 μM. Our results suggest that GBM and NB CSC-like promote the activation of huge quantities of SLRP in response to CSC enrichment, simultaneously acquiring TMZ resistance, cellular heterogeneity, and a quiescent phenotype, suggesting a novel pivotal role for SLRP in drug resistance and cell plasticity of CSC-like, allowing cell survival and ECM/niche modulation potential.

Level of neurotoxic metals in amyotrophic lateral sclerosis: A population-based case-control study

The association between exposure to toxic metals and amyotrophic lateral sclerosis (ALS) was explored in a population-based case-control study in the Sardinia island (Italy), a region characterized by elevated rates of ALS cases. In 34 patients with ALS (mean age, 62 ± 10 years) and 30 controls (mean age, 65 ± 11 years), Al, Cd, Hg, Mn and Pb were determined in blood, hair and urine by sector field inductively coupled mass spectrometry. Results indicated that, in blood, concentrations of Al (p=0.045) and Pb were higher (p=0.026) in ALS patients than in control subjects. In hair, a depletion of Al (p=0.006) and Mn (p=0.032) concentrations in ALS subjects respect to controls was found. In urine, no significant differences between cases and controls were observed. Thus, some metals seemed to be associated with ALS degeneration, but a definitive conclusion is still far considering the multiple risk factors (genetic mutations, environmental toxicants and stressors) involved in the disease. Finally, the interpretation that deregulated metal concentrations can be a consequence of the degenerative process, rather than a cause, is also valid.

AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals.

Immune compatible cystine-functionalized superparamagnetic iron oxide nanoparticles as vascular contrast agents in ultrasonography

Superparamagnetic iron oxide nanoparticles (SPIONs) have been extensively investigated for many biomedical applications. A good quality functionalization that combines imaging goals with a high-level of biocompatibility remains one of the challenges for particle translation into medical practice. Here, we focus on a new functionalization of SPIONs with cystine (Cy-SPIONs). Cystine is able to make SPIONs stable and dispersible in water and in culture cell media. New insights are provided into the biological and immune effects of Cy-SPIONs with a wide variety of standard and molecular assays to evaluate cytotoxicity, cell activation, cytokine release and the expression of 84 genes related to immune responses. A good immune biocompatibility of Cy-SPIONs on primary immune cells was found. The great potential of Cy-SPIONs for further in vivo studies and as contrast agents for magnetic resonance imaging (MRI) is highlighted. In addition, we also exploited ultrasonography, since it is a safer, less expensive and common imaging technology. The good echogenic properties of Cy-SPIONs in water and in whole blood are shown, both in vitro and in a phantom vein for bloodstream simulations. Our results open up a new scenario for future applications of cystine-functionalized SPIONs as immune-compatible ultrasound and MRI contrast agents.

Essential trace elements in amyotrophic lateral sclerosis (ALS): Results in a population of a risk area of Italy

Sardinian (Italy) island population has a uniquely high incidence of amyotrophic lateral sclerosis (ALS). Essential trace element levels in blood, hair, and urine of ALS Sardinian patients were investigated in search of valid biomarkers to recognize and predict ALS. Six elements (Ca, Cu, Fe, Mg, Se, and Zn) were measured in 34 patients compared to 30 age- and sex-matched healthy controls by a validated method. Levels of Ca and Cu in blood and of Se and Zn in hair were significantly higher in ALS than in controls, while urinary excretion of Mg and Se was significantly decreased. The selected cut-off concentrations for these biomarkers may distinguish patients with or without ALS with sufficient sensitivity and specificity. Many positive (as Se-Cu and Se-Zn) and negative associations (as Ca-Mg and Ca-Zn) between elements suggested that multiple metals involved in multiple mechanisms have a role in the ALS degeneration.

Trace elements in ALS patients and their relationships with clinical severity

An exploratory study of trace elements in ALS and their relationships with clinical severity was detected. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that causes irreversible damage in humans, with the consequent loss of function of motoneurons (MNs), with a prognosis up to 5 years after diagnosis. Except to genetic rare cases it is not known the etiology of the disorder. Aim of our research is to investigate the possible role of heavy metals in the severity of the disease. In this study, by the use of plasma mass (ICP-MS), we have analyzed the content of essential and heavy metals such: Pb, Cd, Al, Hg, Mn, Fe, Cu, Zn, Se, Mg, and Ca, in blood, urine and hair of ALS patients and controls; moreover we divided the patients in two groups for disease severity and analyzed the difference among the groups, in order to study a possible involvement of metals in the severity of the damage. Our results suggest a protective role of Selenium, involved in protective antioxidant mechanisms, and a risk factor in the case of presence of Lead in blood. The levels of the other metals are not easy to interpret, because these may be due to life style and for essential metals a consequence of the disease condition, not a cause.

Metals in bones of the middle-aged inhabitants of Sardinia island (Italy) to assess nutrition and environmental exposure

Metals in bones of 72 subjects lived between the twelfth and eighteenth century AC and collected in four Sardinian (Italian insular region) burial sites (Alghero, Bisarcio, Geridu, and Sassari) were determined and used as biomarkers to evaluate diet and potential social-environmental differences. Concentrations of Ba, Ca, Cd, Cu, Hg, Pb, Sr, and Zn were quantified in different types of compact bone (femur, fibula, humerus, radius, tibia, ulna) by sector field inductively coupled plasma mass spectrometry previous acidic digestion and differences among the various burial sites, centuries, types of bone, gender, and age were explored by univariate and multivariate analyses. Results indicated differences between sites in terms of diet: Bisarcio (inland village) had increased ratios of Ba/Ca and Zn/Ca due to higher incidence of vegetables, cereals, and animal foods in the diet; Geridu (coastal village) showed increased Sr/Ca ratio indicating foods of plant and marine origin that were predominant; Alghero (coastal site) and Sassari (inland site) displayed prevalently a mixed diet reflecting a higher economy and food imports. In addition, these latter sites showed increased levels of Hg/Ca (fish, drugs, cosmetics) and Pb/Ca (coins, utensils, pipeline for water). In conclusion, the elemental Ba/Ca, Sr/Ca, and Zn/Ca ratios were indicative of provenance and diet, while Hg/Ca and Pb/Ca ratios were associated to various forms of environmental exposure.

Cancer stem cells and miRNA in the early diagnosis of colorectal carcinoma

Colorectal cancer represents the third cause of death for malignant tumor by incidence and mortality. Evidences suggest that tumor initiation, growth, invasion and cancer expansion are promoted by a small sub-population of tumor cells, called cancer stem cells (CSCs). On the other hand, MicroRNAs constitute a recently discovered class of small non-coding RNAs (about 22 nucletides) found in plants, animals and some viruses, that play key roles in the regulation of gene expression. An increasing number of studies have identified miRNAs as potential biomarkers for human cancer diagnosis, prognosis and therapeutic targets. However, a real translation of miRNA significance into the clinical practice deserves and needs further investigation. To this end, the aim of this work is to identify the expression of some specific microRNAs of this type of cancer, both in tissues and serum of cancer patients and in cancer stem cells, in order to allow early diagnosis. Therefore, some interesting microRNAs were chosen and their level was detected through amplification with real-time PCR method. A preliminary analysis of results shows that in some patients microRNA 21, 221, 18a, 210, 34a, 10b, 16 are overexpressed, while in others they manifest a lower expression. Instead, the microRNA 31 is always overexpressed. We think that this result is related to the clinical stage of the tumor, because patients with similar clinical stage show the same expression.