Emanuela Mari

Pro-inflammatory gene expression in solid glioblastoma microenvironment and in hypoxic stem cells from human glioblastoma

BackgroundAdaptation to hypoxia and consequent pro-inflammatory gene expression of prostate and breast carcinomas have been implicated in the progression toward cancer malignant phenotype. Only partial data are available for the human tumor glioblastoma multiforme (GBM). The aim of our study was to analyze the hypoxic and pro-inflammatory microenvironment in GBMs and to demonstrate that in a stem/progenitor cell line derived from human glioblastoma (GBM-SCs), hypoxia activates a coordinated inflammatory response, evidencing an invasive and migratory phenotype.MethodsFrom each of 10 human solid glioblastomas, clinically and histopathologically characterized, we obtained three surgical samples taken from the center and the periphery of the tumor, and from adjacent host normal tissue. Molecular and morphological analyses were carried out using quantitative real-time PCR and western blot (WB). GBM stem and differentiated cells were incubated under hypoxic conditions and analyzed for pro-inflammatory gene expression and for invasive/migratory behavior.ResultsA panel of selected representative pro-inflammatory genes (RAGE and P2X7R, COX2, NOS2 and, PTX3) were analyzed, comparing tumor, peritumor and host normal tissues. Tumors containing leukocyte infiltrates (as assessed using CD45 immunohistochemistry) were excluded. Selected genes were overexpressed in the central regions of the tumors (i.e. in the more hypoxic areas), less expressed in peripheral regions, and poorly expressed or absent in adjacent normal host tissues. Western blot analysis confirmed that the corresponding pro-inflammatory proteins were also differently expressed. Hypoxic stem cell lines showed a clear time-dependent activation of the entire panel of pro-inflammatory genes as compared to differentiated tumor cells. Biological assays showed that invasive and migratory behavior was strengthened by hypoxia only in GBM stem cells.ConclusionsIn human solid glioblastoma we have observed a coordinated overexpression of a panel of pro-inflammatory genes as compared to host normal tissue. We have also evidenced a similar pattern of overexpressed genes in GBM-SCs after hypoxic treatment, showing also a gain of invasive and migratory function that was lost when these stem cells differentiated. We suggest that, as has been previously described for prostatic and mammary carcinoma, in human glioblastoma acquisition of a proinflammatory phenotype may be relevant for malignant progression.

Synthesis, anti-Candida activity, and cytotoxicity of new (4-(4-iodophenyl)thiazol-2-yl)hydrazine derivatives.

Novel (4-(4-iodophenyl)-thiazol-2-yl)hydrazine derivatives were assayed for their in vitro anti-Candida activity, compared to topical and systemic antifungal drugs, against twenty-seven clinical isolates. The presence of aliphatic chains or specific heteroaromatic rings on hydrazone moiety at position C2 and a 4-iodophenyl at C4 of the thiazole ring gave a promising inhibitory activity especially against Candida albicans and Candida krusei. The most active compounds have been also evaluated for their cytotoxicity and in association with clotrimazole for anti-Candida activity.

Overexpression of estrogen receptor-α in human papillary thyroid carcinomas studied by laser- capture microdissection and molecular biology.

The expression pattern of estrogen receptor (ER) isoforms in normal and tumor thyroid tissues is still controversial and poor defined, therefore, a more detailed study of the distribution of these molecules is needed. Most discrepancies might be due to the methods utilized. We studied the expression of ER isoforms in human papillary thyroid carcinoma (PTC), in fine‐needle aspiration biopsy‐derived specimens, and in cells, using more accurate techniques, such as laser‐capture microdissection, real‐time quantitative PCR, and Western blot. Laser‐capture microdissection allowed us to isolate homogeneous cell populations from human PTC surgical samples. Tumor, peritumor, or normal host tissue of the same sample were separately dissected and analyzed by RT‐PCR and Western blot. Estrogen receptor‐α mRNA was more expressed in cancer‐microdissected cells from human PTC, as compared with microdissected cells obtained from surrounding normal host tissue (450 vs 12, P = 0.001). A similar pattern was observed with Western blot for the ER‐α protein. By contrast, ER‐β mRNA expression was not detected among the microdissected tissue fractions. Fine‐needle aspiration biopsy‐derived specimens showed a similar expression pattern to ER. Moreover, human PTC cell line BCPAP and cancer stem cells from PTC, analyzed under hypoxic conditions, showed a hypoxia‐driven increase in ER‐α expression. In conclusion, ER‐α might have an important role in human PTC, and its overexpression can be studied in routine needle aspirate as a possible marker of malignancy. (Cancer Sci 2011; 102: 1921–1927)

Synthesis and cytotoxicity of novel (thiazol-2-yl)hydrazine derivatives as promising anti-Candida agents.

Thirty-eight new (4-(4-substituted-phenyl/2,4-disubstituted-phenyl)-thiazol-2-yl)hydrazine derivatives were synthesized in good yield and assayed for their in vitro anti-Candida activity, compared to topical and systemic antifungal drugs, against twenty-two clinical isolates of Candida spp. The concurrent presence of aliphatic chains or cycloaliphatic rings at N1-hydrazine and a 4-methyl/4-methoxyphenyl at C4 position of the thiazole nucleus exhibited an interesting anti-Candida inhibitory activity. Moreover, some of the most active compounds showed synergistic antifungal effects and lower cell toxicity when combined with clotrimazole.

Anti-Candida activity and cytotoxicity of a large library of new N-substituted-1,3-thiazolidin-4-one derivatives.

On the basis of the recent findings about the biological properties of thiazolidinones and taking into account the encouraging results about the antifungal activity of some (thiazol-2-yl)hydrazines, new N-substituted heterocyclic derivatives were designed combining the thiazolidinone nucleus with the hydrazonic portion. In details, 1,3-thiazolidin-4-ones bearing (cyclo)aliphatic or (hetero)aromatic moieties linked to the N1-hydrazine at C2 were synthesized and classified into three series according to the aromatic or bicyclic rings connected to the lactam nitrogen of the thiazolidinone. These molecules were assayed for their anti-Candida effects in reference to the biological activity of the conventional topic (clotrimazole, miconazole, tioconazole) and systemic drugs (fluconazole, ketoconazole, amphotericin B). Finally, we investigated the selectivity against fungal cells by testing the compounds endowed with the best MICs on Hep2 cells in order to assess their cell toxicity (CC50) and we noticed that two derivatives were less cytotoxic than the reference drug clotrimazole. Moreover, a preliminary molecular modelling approach has been performed against lanosterol 14-α demethylase (CYP51A1) to rationalize the activity of the tested compounds and to specify the target protein or enzyme.

High‐mobility group box‐1 (HMGB‐1) and serum soluble receptor for advanced glycation end products (sRAGE) in children affected by vernal keratoconjunctivitis

Vernal keratoconjunctivitis (VKC) is a chronic disease affecting conjunctiva even though the immunopathogenetic mechanisms underlying this inflammation are unclear. The aim of our study is to investigate serum levels of HMGB1 and circulating sRAGE in children affected by VKC before and after treatment with cyclosporine A (CsA) eye drops and in a group of healthy children.

Novel 1,3-thiazolidin-4-one derivatives as promising anti-Candida agents endowed with anti-oxidant and chelating properties

Pursuing our recent outcomes regarding the antifungal activity of N-substituted 1,3-thiazolidin-4-ones, we synthesized thirty-six new derivatives introducing aliphatic, cycloaliphatic and heteroaromatic moieties at N1-hydrazine connected with C2 position of the thiazolidinone nucleus and functionalizing the lactam nitrogen with differently substituted (NO2, NH2, Cl and F) benzyl groups. These compounds were tested to evaluate their minimum inhibitory concentration (MIC) against several clinical Candida spp. with respect to topical and systemic reference drugs (clotrimazole, fluconazole, ketoconazole, miconazole, tioconazole, amphotericin B). Moreover, anti-oxidant properties were also evaluated by using different protocols including free radical scavenging (DPPH and ABTS), reducing power (CUPRAC and FRAP), metal chelating and phosphomolybdenum assays. Moreover, for the most active derivatives we assessed the toxicity (CC50) against Hep2 human cells in order to characterize them as multi-target agents for fungal infections.

High levels of serum prostaglandin E2 in children with osteogenesis imperfecta are reduced by neridronate treatment.

Prostaglandin E2 (PGE2) is an activator of bone remodeling, and increase levels of PGE2 are found in several disorders characterized by chronic inflammation. Bisphosphonates are used in the treatment of osteogenesis imperfecta (OI), an inherited disorder characterized by bone fragility and low bone mass. We evaluated the serum PGE2 (ng/mL) level in 16 children affected by OI (11 with mild and 5 with severe forms) at basal time and during treatment with neridronate. The levels of PGE2 in mild and severe forms were increased at basal time compared with controls (13.14 ± 4.2 versus 0.72 ± 0.05, p < 0.01; 15.1 ± 1.5 versus 0.72 ± 0.05, p < 0.01, respectively) and showed a significant decrease after the second (T1) cycle of treatment (mild: 4.97 ± 5.0 versus 13.14 ± 4.2, p < 0.01; severe: 5.32 ± 4.5 versus 15.1 ± 1.5, p < 0.01) with a further significant decrease after the fourth (T2) cycle. The high basal PGE2 levels in OI, a noninflammatory disorder, could be explained by stress-induced release mediated by inducible cyclooxygenase-2-catalyzed pathway. The reduction obtained by treatment with bisphosphonates could be attributed to a direct pharmacological effect since these drugs has been reported to modulate the release of proinflammatory mediators.

Graphene oxide nanoribbons induce autophagic vacuoles in neuroblastoma cell lines

Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO) nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs) and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2) and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS), mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM) for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells.

Synthesis, biological evaluation and quantitative structure-active relationships of 1,3-thiazolidin-4-one derivatives. A promising chemical scaffold endowed with high antifungal potency and low cytotoxicity

With reference to recent studies reporting on the various biological properties of the thiazolidinone scaffold, we synthesized more than a hundred compounds characterized by a 1,3-thiazolidin-4-one nucleus derivatised at the C2 with a hydrazine bridge linked to (cyclo)aliphatic or hetero(aryl) moieties, and their N-benzylated derivatives. These molecules were assayed as potential anti-Candida agents and they were shown to possess comparable, and in some cases higher biological activity than well-established topical and systemic antimycotic drugs (i.e. clotrimazole, fluconazole, ketoconazole, miconazole, tioconazole, amphotericin B). Compounds endowed with the lowest MICs underwent further testing in order to assess their cytotoxic effect (CC50) on Hep2 cells, which demonstrated their relative safety. Finally, QSAR and 3-D QSAR models were used to predict putative chemical modifications of the 1,3-thiazolidin-4-one scaffold in order to design new and potential more active compounds against Candida spp.