Mimicking the Tumor Niche: Methods for Isolation, Culture, and Characterization of Cancer Stem Cells and Multicellular Spheroids.

Cancer stem cells (CSCs) play a significant role in driving several tumor hallmarks. Their behavior and tumor progression are strictly related to the tumor microenvironment (TME). The dynamic interplay between CSCs and TME drives metastasis, chemoresistance, and disease relapse. In this chapter, we describe different techniques and protocols for isolating, culturing, and characterizing CSCs and we explain the methodology for the culture of multicellular spheroids comprising CSCs.

Toxic Metal and Essential Element Concentrations in the Blood and Tissues of Pancreatic Ductal Adenocarcinoma Patients.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal neoplasm, and it has an average 5-year survival rate of less than 10%. Although the factors that influence PDAC development remain unclear, exposure to toxic metals or the imbalance in essential elements may have a role in PDAC-associated metabolic pathways. METHODS: This study determined the concentrations of Cd, Cr, Cu, Fe, Mn, Ni, Pb, Se and Zn in whole blood, cancer and non-cancer tissues of patients affected by PDAC, and compared them with levels in healthy controls using inductively coupled plasma mass spectrometry. RESULTS: Results of the whole blood showed significantly higher levels of Cr, Cu and Cu/Zn ratio in PDAC patients compared to the controls. In addition, the concentrations of Cu, Se, Fe and Zn significantly increased in cancer tissue compared to the healthy counterparts. CONCLUSIONS: This study revealed evidence of altered metal levels in the blood and pancreatic tissues of PDAC patients with respect to healthy controls. These changes may contribute to multiple mechanisms involved in metal-induced carcinogenesis, including oxidative stress, DNA damage, genetic alteration, decreased antioxidant barriers and inflammatory responses. Thus, the analysis of metals can be used in the diagnosis and monitoring of PDAC neoplasms.

Human tissue lead (Pb) levels and amyotrophic lateral sclerosis: a systematic review and meta-analysis of case-control studies.

AIM: To combine the current scientific literature evidence and elucidate the differences of lead (Pb) bioaccumulation in human tissues by comparing amyotrophic lateral sclerosis (ALS) patients and healthy controls. METHODS: We systematically searched for case-control studies on the association of Pb levels with ALS, in human cells, tissues, and body fluids (nervous tissue, muscle, blood, cerebrospinal fluid, urine, skin appendages). Then, we performed a meta-analysis for all the tissues in which at least five case-control studies were available: whole blood (9 studies), serum/plasma (5 studies), and cerebrospinal fluid (CSF) (6 studies). Differences between cases and controls were evaluated using standardized mean difference, and combined estimates were derived using random effect maximum likelihood (REML) meta-analyses. RESULTS: Among 1734 records, we identified 46 full-text studies, of which 14 case-control studies met the meta-analysis inclusion criteria. We found higher Pb levels in ALS cases than controls in blood (standardized mean difference (SMD) = 0.61; 95% confidence interval (CI) 0.20, 1.01; p = 0.003), plasma/serum (SMD = 0.27; 95% CI - 0.16, 0.70; p = 0.26), and CSF (SMD = 0.53; 95% CI - 0.09, 1.15; p = 0.09). CONCLUSIONS: This work provides further evidence of the association between Pb bioaccumulation and ALS in body fluids. The lack of association studies in solid tissues did not allow a robust meta-analysis. Future prospective studies are needed to clarify the causality in the association of Pb bioaccumulation with ALS.

The levels of trace elements in sputum as biomarkers for idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a rare lung disease that quickly leads to death. This paper addressed the issue of whether the levels of trace elements in sputum samples are suitable biomarkers for IPF disease. The sputum Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn concentrations were measured by sector field inductively coupled plasma mass spectrometry in populations sampled in Sardinia Island (Italy) including 31 patients with IPF, 31 patients with other lung-related diseases and 30 age- and gender-matched healthy controls. Risk factors in the disease as gender, age, severity and duration of the disease were assessed. Results showed that IPF patients had significantly increased sputum levels of Cd, Cr, Cu and Pb respect to controls. In males, but not in females, sputum levels of Cd, Cr and Cu were significantly higher in IPF cases respect to controls. In addition, Cr and Pb were increased in male patients with IPF compared to male patients with other lung diseases. Regarding Zn, it was found higher with the more serious stage of disease. Moreover, the ratios Cu/Zn, Fe/Mn and Cu/Mn were significantly increased in IPF patients and in non-IPF patients than in control subjects. These data showed clear increases in the concentration of some trace elements in sputum from patients with IPF and patients with other lung-related diseases that may contribute to the injury. The non-invasiveness of the sputum analysis is beneficial for its use as biomarker of trace element status in diseased patients for both the researcher and the clinic.

An overview on amyotrophic lateral sclerosis and cadmium.

The present review represents an update about the knowledge of the possible role of Cadmium (Cd) in amyotrophic lateral sclerosis (ALS) initiation and its progression. ALS is a neurodegenerative disease that occurs in adulthood; its etiology is unknown and leads to death within a few years from its appearance. Among the various possible causes that can favor the development of the disease, heavy metals cannot be excluded. Cadmium is a heavy metal that does not play a biological role, but its neurotoxicity is well known. Numerous in vitro studies on cell and animal models confirm the toxicity of the metal on the nervous system, but these data are not accompanied by an epidemiological evidence, and, thus, an unclear correlation between Cd and the onset of the disease can be pointed out. On the other hand, a possible multifactorial and synergic mechanism in which Cd may have a role can explain the ALS onset. More efforts in new clinical, biochemical, and epidemiological studies are necessary to better elucidate the involvement of Cd in this lethal disease.

The Inhibitory Role of miR-486-5p on CSC Phenotype Has Diagnostic and Prognostic Potential in Colorectal Cancer.

Colorectal cancer (CRC) is the third most frequent cancer worldwide and the second cause of cancer deaths. Increasing evidences supports the idea that the poor prognosis of patients is related to the presence of cancer stem cells (CSCs), a cell population able to drive cancer recurrence and metastasis. The deregulation of microRNAs (miRNAs) plays a role in the formation of CSC. We investigated the role of hsa-miR-486-5p (miR-486-5p) in CRC, CSCs, and metastasis, in order to reach a better understanding of the biomolecular and epigenetic mechanisms mir-486-5p-related. The expression of miR-486-5p was investigated in three different matrices from CRC patients and controls and in CSCs obtained from the CRC cell lines HCT-116, HT-29, and T-84. In the human study, miR-486-5p was up-regulated in serum and stool of CRC patients in comparison with healthy controls but down-regulated in tumor tissue when compared with normal mucosa. miR-486-5p was also down-regulated in the sera of metastatic patients. In vitro, miR-486-5p was down-regulated in CSC models and it induced an inhibitory effect on stem factors and oncogenes in the main pathways of CSCs. Our results provide a step forward in understanding the role of mir-486-5p in CRC and CSC, and suggest that further studies are needed to investigate its diagnostic and prognostic power, possibly in combination with other biomarkers.

Amyotrophic lateral sclerosis and lead: A systematic update.

Heavy metals are considered to be among the leading environmental factors that trigger amyotrophic lateral sclerosis (ALS). However, no convincing biopathological mechanism and therapeutic clinical implication of such metals in ALS pathogenesis have been established. This is partly attributable to the technical and scientific difficulties in demonstrating a direct and causative role of heavy metals in the onset of ALS in patients. However, a body of epidemiological, clinical and experimental evidences suggest that lead (Pb), more than other metals, could actually play a major role in the onset and progression of ALS. Here, to clarify the nature of the association and the causative role of Pb in ALS, we comprehensively reviewed the scientific literature of the last decade with objective database searches and the methods typically adopted in systematic reviews, critically analysing and summarising the various scientifically sound evidence on the relationship between ALS and Pb. From these tasks, we noted a number of multidisciplinary associations between ALS and Pb, and specifically the importance of occupational exposure to Pb in ALS development and/or progression. We also report the possible involvement of TAR DNA binding protein (TDP-43)-based molecular mechanism in Pb-mediated ALS, although these data rely on a single study, which included both in vitro experiments and an animal model, and are therefore still preliminary. Finally, we briefly examined whether this knowledge could inspire new targeted therapies and policies in the fight against ALS.

A pilot study to evaluate the levels of aqueous humor trace elements in open-angle glaucoma.

BACKGROUND: Primary open angle glaucoma (POAG) is the most common form of chronic, progressive optic neuropathies characterized by slow degeneration of the retinal ganglion cells and their axons, resulting in visual field loss. Risk factors for this disease are elevated intraocular pressure (IOP), increased age, European and African ethnicity, family history, myopia and decreased corneal thickness. In addition, studies indicated that levels of trace elements are also significantly related to the POAG. METHOD: The association between toxic and essential elements and POAG was explored in a population-based case-control study in the Sardinia Island (Italy). The aqueous humor levels of Al, Cd, Cu, Fe, Hg, Mn, Ni, Pb and Zn were measured in 25 POAG patients compared to 20 age- and gender-matched healthy controls by sector field inductively coupled mass spectrometry. Risk factors as gender, age and increased IOP were also explored. RESULTS: The concentrations of Fe, Hg and Zn were significantly higher in POAG patients than in control subjects, showing these elements as possible determinants in POAG development or degeneration. Other findings were the increased Cu and Fe levels in glaucomatous patients with age less than 70 years. Levels of Ni were found elevated in POAG females. Mercury accumulated more in POAG females, in patients over 70 years and in those with higher levels of IOP in the left eye. Moreover, the positive associations CuFe and Mn-Zn may indicate synergistic effects of elements. CONCLUSIONS: Altogether, these findings suggested a multifactorial role in the risk for POAG disease. The present study documented the levels of trace elements in aqueous humor of Sardinian POAG patients for the first time.

Deregulation of cancer-stem-cell-associated miRNAs in tissues and sera of colorectal cancer patients.

Colorectal cancer (CRC) is a deadly tumour in Western countries characterized by high cellular/molecular heterogeneity. Cancer stem cells (CSC) act in cancer recurrence, drug-resistance and in metastatic epithelial-to-mesenchymal transition. microRNAs (miRNAs) contribute to cancer is increasing, and miRNA roles in CSC phenotype and fate and their utility as CRC biomarkers have also been reported. Here, we investigated miR-21, miR-221, miR-18a, miR-210, miR-31, miR-34a, miR-10b and miR-16 expression in experimental ALDH+ and CD44+/CD326+ colorectal CSCs obtained from the human CRC cell lines HCT-116, HT-29 and T-84. Then, we moved our analysis in cancer tissue (CT), healthy tissue (HT) and serum (S) of adult CRC patients (n=12), determining relationships with clinical parameters (age, sex, metastasis, biochemical serum markers). Specific miRNA patterns were evident in vitro (normal, monolayers and CSCs) and in patients' samples stratified by TNM stage (LOW vs HIGH) or metastasis (Met vs no-Met). miR-21, miR-210, miR-34a upregulation ad miR-16 dowregulation associated with the CSCs phenotype. miR-31b robustly overexpressed in monolayers and CSCs, and in CT ad S of HIGH grade and Met patients, suggesting a role as marker of CRC progression and metastasis. miR-18a upregulated in all cancer models and associated to CSC phenotype, and to metastasis and age in patients. miR-10b downregulated in CT and S of LOW/HIGH grade and no-Met patients. Our results identify miRNAs useful as colorectal CSC biomarker and that miR-21, miR-210, miR-10b and miR-31b are promising markers of CRC. A specific role of miR-18a as metastatic CRC serum biomarker in adult patients was also highlighted.

Agenesis of the posterior arch of the atlas and complex alterations of the craniovertebral junction: A case report.

The craniovertebral junction is a unique part of the somite-derived axial skeleton. The absence or hypoplasia of the posterior arch of C1 is frequently associated with compensatory hypertrophy of the anterior arch of C1 and of the spinous process of C2. Here, we report a patient with agenesis of the posterior arch of C1 without neurologic deficits. Our patient presented with complex alterations of the craniovertebral junction that involved interactions between the condyles, clivus, atlas, and epistropheus. To our knowledge, dislocation of the odontoid process above the Chamberlain line, including cranial migration of the anterior arch of C1, has not been reported in the literature.

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.

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.

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.

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.

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.

Blood metals concentration in type 1 and type 2 diabetics.

Mechanisms for the onset of diabetes and the development of diabetic complications remain under extensive investigations. One of these mechanisms is abnormal homeostasis of metals, as either deficiency or excess of metals, can contribute to certain diabetic outcomes. Therefore, this paper will report the blood levels of chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), mercury (Hg), nickel (Ni), lead (Pb), selenium (Se), and zinc (Zn) in subjects with type 1 diabetes (n = 192, mean age 48.8 years, mean disease duration 20.6 years), type 2 diabetes (n = 68, mean age 68.4 years, mean disease duration 10.2 years), and in control subjects (n = 59, mean age 57.2 years), and discuss the results indicating their possible role in diabetes. The metal concentrations were measured by sector field inductively coupled plasma mass spectrometry after microwave-induced acid digestion of blood samples. The accuracy was checked using a blood-based certified reference material, and recoveries of all elements were in the range of 92-101 % of certified values. Type 1 diabetes was found to be associated with Cr (p = 0.02), Mn (p < 0.001), Ni (p < 0.001), Pb (p = 0.02), and Zn (p < 0.001) deficiency, and type 2 diabetes with Cr (p = 0.014), Mn (p < 0.001), and Ni (p < 0.001) deficiency. These deficiencies were appreciated also subdividing the understudied patients for gender and age groups. Furthermore, in type 1 diabetes, there was a positive correlation between Pb and age (p < 0.001, ρ = 0.400) and Pb and BMI (p < 0.001, ρ = 0.309), while a negative correlation between Fe and age (p = 0.002, ρ = -0.218). In type 2 diabetes, there was a negative correlation between Fe and age (p = 0.017, ρ = -0.294) and Fe and BMI (p = 0.026, ρ = -0.301). Thus, these elements may play a role in both forms of diabetes and combined mineral supplementations could have beneficial effects.

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.

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.

Cytoskeletal proteins in the cerebrospinal fluid as biomarker of multiple sclerosis.

The axonal cytoskeleton is a finely organized system, essential for maintaining the integrity of the axon. Axonal degeneration is implicated in the pathogenesis of unremitting disability of multiple sclerosis (MS). Purpose of this study is to evaluate levels of cytoskeletal proteins such as neurofilament light protein (NFL), glial fibrillary acidic protein (GFAP), and ß-tubulin (ß-Tub) isoforms II and III in the cerebrospinal fluid (CSF) of MS patients and their correlation with MS clinical indices. CSF levels of cytoskeletal proteins were determined in 51 patients: 33 with MS and 18 with other neurological diseases (OND). NFL, GFAP and ß-Tub II proteins were significantly higher (p < 0.0001) in MS than in OND group; no significant difference (p > 0.05) was found between MS and OND with regard to ß-Tub III. Interestingly, levels of ß-Tub III and NFL were higher in progressive than in remitting MS forms; on the contrary, higher levels of ß-Tub II and GFAP were found in remitting MS forms. However, with the exception of ß-Tub III, all proteins tend to decrease their CSF levels concomitantly with the increasing disability (EDSS) score. Overall, our results might indicate ß-Tub II as a potential candidate for diagnostic and ß-Tub III as a possible prognostic biomarker of MS. Therefore, further analyses are legitimated and desirable.