Rossella Solmi

Osteoinduction by Chitosan-Complexed BMP: Morpho-Structural Responses in an Osteoporotic Model

Bone quality is the result of a complex relationship between bone mass, bone structure, and mechanical characteristics of these individual components. The mass of bone tissue is affected by nutritional factors and other causes, such as bone growth factors like bone morphogenetic proteins (BMPs). Since chitosans promote ordered regeneration of soft tissue and osteoinduction, an osteoporotic model was studied to evaluate the pattern of bone regeneration in the presence of BMP linked to chitosan. BMP was released from the chitosan matrix as a consequence of chitosan biodegradation. Our data show that the association of BMP with chitosan seemed to improve the bone tissue regeneration in a surgical bone defect. This result provides validity to biochemical approaches for therapeutical correction of afflictions in the elderly, such as osteoporosis.

Diurnal suppression of EGFR signalling by glucocorticoids and implications for tumour progression and treatment

Signal transduction by receptor tyrosine kinases (RTKs) and nuclear receptors for steroid hormones is essential for body homeostasis, but the cross-talk between these receptor families is poorly understood. We observed that glucocorticoids inhibit signalling downstream of EGFR, an RTK. The underlying mechanism entails suppression of EGFR’s positive feedback loops and simultaneous triggering of negative feedback loops that normally restrain EGFR. Our studies in mice reveal that the regulation of EGFR’s feedback loops by glucocorticoids translates to circadian control of EGFR signalling: EGFR signals are suppressed by high glucocorticoids during the active phase (night-time in rodents), while EGFR signals are enhanced during the resting phase. Consistent with this pattern, treatment of animals bearing EGFR-driven tumours with a specific kinase inhibitor was more effective if administered during the resting phase of the day, when glucocorticoids are low. These findings support a circadian clock-based paradigm in cancer therapy.

Qualitative assessment of natural apatite in vitro and in vivo.

Among the natural and synthetic materials investigated as bone graft substitutes, much interest has been focused on natural apatite obtained from low temperature heat-deproteinated compact bone. Previous research demonstrates that, when treated at a temperature below 500 degrees C, this material maintains its characteristic ultrastructural features, with a high surface/volume ratio, while as an implant material, it offers the host tissue a large surface of interaction. In vitro and in vivo tests showed that natural apatite is well tolerated and is a good osteoconducing material. The present in vivo study in rabbits was carried out to first investigate the behavior and capacity of natural apatite implants to stimulate bone ingrowth, and then to analyze the cells located at the bone/material interface. Synthetic hydroxyapatite was used as a control material. In a parallel in vitro study, we investigated the activity of differentiated osteoblasts and periosteal cells obtained from rats and new-born rabbits, incubated with natural apatite and synthetic hydroxyapatite. The in vivo study showed that natural apatite allows osteoblasts to form new bone tissue, adhering to the implant with ingrowth into the implant structure. In the presence of synthetic hydroxyapatite, a less pronounced osteoblastic activity was observed. In agreement with these observations, the in vitro study showed that natural apatite is more effective in attracting cells, favoring their proliferation and stimulating alkaline phosphatase activity. These findings suggest that natural apatite is more suitable for bone filling or bone regeneration than synthetic hydroxyapatite.

Microarray-based identification and RT-PCR test screening for epithelial-specific mRNAs in peripheral blood of patients with colon cancer.

BackgroundThe efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions.MethodsIn this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22) that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription – polymerase chain reaction (RT-PCR).ResultsOur present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify.ConclusionThe design of new approaches to identify such markers is warranted.

Interactions of fibroblasts with soldered and laser-welded joints

Relatively little is known about the biocompatibility of the soldered or laser-welded joints of dental appliances. We investigated the reaction of human gingival fibroblasts cultured in vitro in direct contact with samples of soldered and laser-welded joints from orthodontic lingual arches. Contrast phase light microscopy was used to evaluate cell adhesion, morphology and proliferation after 6 and 24h and after 7 and 16 days. Scanning electron microscopy (SEM) was performed at 16 days. Our in vitro findings provide evidence that laser-welded orthodontic appliances have superior fibroblast biocompatibility.

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines.

BackgroundEGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF.MethodsCell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A.ResultsCaco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment.In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases.ConclusionThis is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cyto-morphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death.

A role for epidermal growth factor receptor in idiopathic pulmonary fibrosis onset

In idiopathic pulmonary fibrosis (IPF) patients the presence of missense polymorphisms (SNP) in members of the epidermal growth factor receptor (EGFR) family or their genetic association could influence the binding affinity of natural ligands, modifying the expression and the behavior of the correlated genes. EGFR family members are particularly involved in the epithelial injury and fibrotic process in IPF. Genetic variations in HER family of receptors may alter the possible therapeutic efficacy of EGFR inhibitors. This study aimed to analyze the relationships between IPF and specific EGF receptor family functional polymorphisms. We tested the presence of common EGFR, HER2 and HER3 non-synonymous SNPs in the peripheral blood of 20 Italian IPF patients and their association with the disease. Our data indicated that the HER2 variant allele frequency was significantly lower in patients than in controls, with an odds ratio of 0.31 (95% CI 0.080, 0.98). Our finding suggests that HER2 variant could be a protective factor against IPF onset.

Adenomatous polyposis coli (APC) regulates miR17-92 cluster through β-catenin pathway in colorectal cancer.

Adenomatous polyposis coli (APC) mutation is the most common genetic change in sporadic colorectal cancer (CRC). Although deregulations of miRNAs have been frequently reported in this malignancy, APC-regulated miRNAs have not been extensively documented. Here, by using an APC-inducible cell line and array analysis, we identified a total of 26 deregulated miRNAs. Among them, members of miR-17-92 cluster were dramatically inhibited by APC and induced by enforced expression of β-catenin. Furthermore, we demonstrate that activated β-catenin resulted from APC loss binds to and activates the miR-17-92 promoter. Notably, enforced expression of miR-19a overrides APC tumor suppressor activity, and knockdown of miR-19a in cancer cells with compromised APC function reduced their aggressive features in vitro. Finally, we observed that expression of miR-19a significantly correlates with β-catenin levels in colorectal cancer specimens, and it is associated to the aggressive stage of tumor progression. Thus, our study reveals that miR-17-92 cluster is directly regulated by APC/β-catenin pathway and could be a potential therapeutic target in colon cancers with aberrant APC/β-catenin signaling.

A candidate gene study of one-carbon metabolism pathway genes and colorectal cancer risk.

The risk of colorectal cancer (CRC) may be influenced by aberrant DNA methylation and altered nucleotide synthesis and repair, possibly caused by impaired dietary folate intake as well as by polymorphic variants in one-carbon metabolism genes. A case-control study using seventy-one CRC patients and eighty unrelated healthy controls was carried out to assess the genetic association of fifteen SNP and one insertion in nine genes belonging to the folate pathway. Polymorphism selection was based on literature data, and included those which have a known or suspected functional impact on cancer and missense polymorphisms that are most likely to alter protein function. Genotyping was performed by real-time PCR and PCR followed by restriction analysis. The likelihood ratio statistic indicated that most of the polymorphisms were not associated with the risk of CRC. However, an increased risk of CRC was observed for two variant alleles of SNP mapping on the transcobalamin 2 gene (TCN2): C776G (rs1801198) and c.1026-394T>G (rs7286680). Considering the crucial biological function played by one-carbon metabolism genes, further investigations with larger cohorts of CRC patients are needed in order to confirm our preliminary results. These preliminary results indicate that TCN2 polymorphisms can be a susceptibility factor for CRC.

A module of inflammatory cytokines defines resistance of colorectal cancer to EGFR inhibitors

Epidermal Growth Factor Receptor (EGFR) activates a robust signalling network to which colon cancer tumours often become addicted. Cetuximab, one of the monoclonal antibodies targeting this pathway, is employed to treat patients with colorectal cancer. However, many patients are intrinsically refractory to this treatment, and those who respond develop secondary resistance along time. Mechanisms of cancer cell resistance include either acquisition of new mutations or non genomic activation of alternative signalling routes. In this study, we employed a colon cancer model to assess potential mechanisms driving resistance to cetuximab. Resistant cells displayed increased ability to grow in suspension as colonspheres and this phenotype was associated with poorly organized structures. Factors secreted from resistant cells were causally involved in sustaining resistance, indeed administration to parental cells of conditioned medium collected from resistant cells was sufficient to reduce cetuximab efficacy. Among secreted factors, we report herein that a signature of inflammatory cytokines, including IL1A, IL1B and IL8, which are produced following EGFR pathway activation, was associated with the acquisition of an unresponsive phenotype to cetuximab in vitro. This signature correlated with lack of response to EGFR targeting also in patient-derived tumour xenografts. Collectively, these results highlight the contribution of inflammatory cytokines to reduced sensitivity to EGFR blockade and suggest that inhibition of this panel of cytokines in combination with cetuximab might yield an effective treatment strategy for CRC patients refractory to anti-EGFR targeting.