Glut-3 Gene Knockdown as a Potential Strategy to Overcome Glioblastoma Radioresistance.

The hypoxic pattern of glioblastoma (GBM) is known to be a primary cause of radioresistance. Our study explored the possibility of using gene knockdown of key factors involved in the molecular response to hypoxia, to overcome GBM radioresistance. We used the U87 cell line subjected to chemical hypoxia generated by CoCl2 and exposed to 2 Gy of X-rays, as single or combined treatments, and evaluated gene expression changes of biomarkers involved in the Warburg effect, cell cycle control, and survival to identify the best molecular targets to be knocked-down, among those directly activated by the HIF-1α transcription factor. By this approach, glut-3 and pdk-1 genes were chosen, and the effects of their morpholino-induced gene silencing were evaluated by exploring the proliferative rates and the molecular modifications of the above-mentioned biomarkers. We found that, after combined treatments, glut-3 gene knockdown induced a greater decrease in cell proliferation, compared to pdk-1 gene knockdown and strong upregulation of glut-1 and ldha, as a sign of cell response to restore the anaerobic glycolysis pathway. Overall, glut-3 gene knockdown offered a better chance of controlling the anaerobic use of pyruvate and a better proliferation rate reduction, suggesting it is a suitable silencing target to overcome radioresistance.

Biological and Mechanical Characterization of the Random Positioning Machine (RPM) for Microgravity Simulations.

The rapid improvement of space technologies is leading to the continuous increase of space missions that will soon bring humans back to the Moon and, in the coming future, toward longer interplanetary missions such as the one to Mars. The idea of living in space is charming and fascinating; however, the space environment is a harsh place to host human life and exposes the crew to many physical challenges. The absence of gravity experienced in space affects many aspects of human biology and can be reproduced in vitro with the help of microgravity simulators. Simulated microgravity (s-µg) is applied in many fields of research, ranging from cell biology to physics, including cancer biology. In our study, we aimed to characterize, at the biological and mechanical level, a Random Positioning Machine in order to simulate microgravity in an in vitro model of Triple-Negative Breast Cancer (TNBC). We investigated the effects played by s-µg by analyzing the change of expression of some genes that drive proliferation, survival, cell death, cancer stemness, and metastasis in the human MDA-MB-231 cell line. Besides the mechanical verification of the RPM used in our studies, our biological findings highlighted the impact of s-µg and its putative involvement in cancer progression.

Hypoxia Transcriptomic Modifications Induced by Proton Irradiation in U87 Glioblastoma Multiforme Cell Line.

In Glioblastoma Multiforme (GBM), hypoxia is associated with radioresistance and poor prognosis. Since standard GBM treatments are not always effective, new strategies are needed to overcome resistance to therapeutic treatments, including radiotherapy (RT). Our study aims to shed light on the biomarker network involved in a hypoxic (0.2% oxygen) GBM cell line that is radioresistant after proton therapy (PT). For cultivating cells in acute hypoxia, GSI's hypoxic chambers were used. Cells were irradiated in the middle of a spread-out Bragg peak with increasing PT doses to verify the greater radioresistance in hypoxic conditions. Whole-genome cDNA microarray gene expression analyses were performed for samples treated with 2 and 10 Gy to highlight biological processes activated in GBM following PT in the hypoxic condition. We describe cell survival response and significant deregulated pathways responsible for the cell death/survival balance and gene signatures linked to the PT/hypoxia configurations assayed. Highlighting the molecular pathways involved in GBM resistance following hypoxia and ionizing radiation (IR), this work could suggest new molecular targets, allowing the development of targeted drugs to be suggested in association with PT.

Molecular Investigation on a Triple Negative Breast Cancer Xenograft Model Exposed to Proton Beams.

Specific breast cancer (BC) subtypes are associated with bad prognoses due to the absence of successful treatment plans. The triple-negative breast cancer (TNBC) subtype, with estrogen (ER), progesterone (PR) and human epidermal growth factor-2 (HER2) negative receptor status, is a clinical challenge for oncologists, because of its aggressiveness and the absence of effective therapies. In addition, proton therapy (PT) represents an effective treatment against both inaccessible area located or conventional radiotherapy (RT)-resistant cancers, becoming a promising therapeutic choice for TNBC. Our study aimed to analyze the in vivo molecular response to PT and its efficacy in a MDA-MB-231 TNBC xenograft model. TNBC xenograft models were irradiated with 2, 6 and 9 Gy of PT. Gene expression profile (GEP) analyses and immunohistochemical assay (IHC) were performed to highlight specific pathways and key molecules involved in cell response to the radiation. GEP analysis revealed in depth the molecular response to PT, showing a considerable immune response, cell cycle and stem cell process regulation. Only the dose of 9 Gy shifted the balance toward pro-death signaling as a dose escalation which can be easily performed using proton beams, which permit targeting tumors while avoiding damage to the surrounding healthy tissue.

Nutraceutical Compounds as Sensitizers for Cancer Treatment in Radiation Therapy.

The improvement of diagnostic techniques and the efficacy of new therapies in clinical practice have allowed cancer patients to reach a higher chance to be cured together with a better quality of life. However, tumors still represent the second leading cause of death worldwide. On the contrary, chemotherapy and radiotherapy (RT) still lack treatment plans which take into account the biological features of tumors and depend on this for their response to treatment. Tumor cells' response to RT is strictly-connected to their radiosensitivity, namely, their ability to resist and to overcome cell damage induced by ionizing radiation (IR). For this reason, radiobiological research is focusing on the ability of chemical compounds to radiosensitize cancer cells so to make them more responsive to IR. In recent years, the interests of researchers have been focused on natural compounds that show antitumoral effects with limited collateral issues. Moreover, nutraceuticals are easy to recover and are thus less expensive. On these bases, several scientific projects have aimed to test also their ability to induce tumor radiosensitization both in vitro and in vivo. The goal of this review is to describe what is known about the role of nutraceuticals in radiotherapy, their use and their potential application.

Proton Therapy and Src Family Kinase Inhibitor Combined Treatments on U87 Human Glioblastoma Multiforme Cell Line.

Glioblastoma Multiforme (GBM) is the most common of malignant gliomas in adults with an exiguous life expectancy. Standard treatments are not curative and the resistance to both chemotherapy and conventional radiotherapy (RT) plans is the main cause of GBM care failures. Proton therapy (PT) shows a ballistic precision and a higher dose conformity than conventional RT. In this study we investigated the radiosensitive effects of a new targeted compound, SRC inhibitor, named Si306, in combination with PT on the U87 glioblastoma cell line. Clonogenic survival assay, dose modifying factor calculation and linear-quadratic model were performed to evaluate radiosensitizing effects mediated by combination of the Si306 with PT. Gene expression profiling by microarray was also conducted after PT treatments alone or combined, to identify gene signatures as biomarkers of response to treatments. Our results indicate that the Si306 compound exhibits a radiosensitizing action on the U87 cells causing a synergic cytotoxic effect with PT. In addition, microarray data confirm the SRC role as the main Si306 target and highlights new genes modulated by the combined action of Si306 and PT. We suggest, the Si306 as a new candidate to treat GBM in combination with PT, overcoming resistance to conventional treatments.

Proton-irradiated breast cells: molecular points of view.

Breast cancer (BC) is the most common cancer in women, highly heterogeneous at both the clinical and molecular level. Radiation therapy (RT) represents an efficient modality to treat localized tumor in BC care, although the choice of a unique treatment plan for all BC patients, including RT, may not be the best option. Technological advances in RT are evolving with the use of charged particle beams (i.e. protons) which, due to a more localized delivery of the radiation dose, reduce the dose administered to the heart compared with conventional RT. However, few data regarding proton-induced molecular changes are currently available. The aim of this study was to investigate and describe the production of immunological molecules and gene expression profiles induced by proton irradiation. We performed Luminex assay and cDNA microarray analyses to study the biological processes activated following irradiation with proton beams, both in the non-tumorigenic MCF10A cell line and in two tumorigenic BC cell lines, MCF7 and MDA-MB-231. The immunological signatures were dose dependent in MCF10A and MCF7 cell lines, whereas MDA-MB-231 cells show a strong pro-inflammatory profile regardless of the dose delivered. Clonogenic assay revealed different surviving fractions according to the breast cell lines analyzed. We found the involvement of genes related to cell response to proton irradiation and reported specific cell line- and dose-dependent gene signatures, able to drive cell fate after radiation exposure. Our data could represent a useful tool to better understand the molecular mechanisms elicited by proton irradiation and to predict treatment outcome.

Radiation Gene-expression Signatures in Primary Breast Cancer Cells.

BACKGROUND/AIM: In breast cancer (BC) care, radiation therapy (RT) is an efficient treatment to control localized tumor. Radiobiological research is needed to understand molecular differences that affect radiosensitivity of different tumor subtypes and the response variability. The aim of this study was to analyze gene expression profiling (GEP) in primary BC cells following irradiation with doses of 9 Gy and 23 Gy delivered by intraoperative electron radiation therapy (IOERT) in order to define gene signatures of response to high doses of ionizing radiation. MATERIALS AND METHODS: We performed GEP by cDNA microarrays and evaluated cell survival after IOERT treatment in primary BC cell cultures. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to validate candidate genes. RESULTS: We showed, for the first time, a 4-gene and a 6-gene signature, as new molecular biomarkers, in two primary BC cell cultures after exposure at 9 Gy and 23 Gy respectively, for which we observed a significantly high survival rate. CONCLUSION: Gene signatures activated by different doses of ionizing radiation may predict response to RT and contribute to defining a personalized biological-driven treatment plan.

DVWA gene polymorphisms and osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a degenerative joints disorder influenced by genetic predisposition. We reported that rs11718863 DVWA SNP was represented in Sicilian with a more severe Kellgren and Lawrence (KL) radiographic grade, displaying its predictive role as OA marker progression. Here, we describe the DVWA SNPs: rs11718863, rs7639618, rs7651842, rs7639807 and rs17040821 probably able to induce protein functional changes. FINDINGS: Sixty-one Sicilian patients with knee OA and 100 healthy subjects were enrolled. Clinical and radiographic evaluation was performed using AKSS scores and KL. Linkage Disequilibrium (LD) analyses were performed in order to verify whether the SNPs segregate as haplotype. All DVWA SNPs'MinorAllele Frequencies (MAF) were greater than in the European. The rs7639618 SNP showed a statistical association with KL. Our analyses show that a LD exists among rs11718863 and rs7639618, as well as between rs7651842, rs7639807 and rs17040821 SNPs. We also observed that three out of the 161 individuals investigated were simultaneously homozygous carriers of the rs7651842, rs7639807 and rs17040821 MAF alleles. CONCLUSIONS: In summary, the purpose of this preliminary research was to highlight possible associations between DVWA SNPs and OA clinical and radiographic data. This work represents a multidisciplinary medicine approach to study OA where clinical, radiological and genetic evaluation could contribute to better define OA grading.

Theoretical and experimental study of the role of cell-cell dipole interaction in dielectrophoretic devices: application to polynomial electrodes.

BACKGROUND: We aimed to investigate the effect of cell-cell dipole interactions in the equilibrium distributions in dielectrophoretic devices. METHODS: We used a three dimensional coupled Monte Carlo-Poisson method to theoretically study the final distribution of a system of uncharged polarizable particles suspended in a static liquid medium under the action of an oscillating non-uniform electric field generated by polynomial electrodes. The simulated distributions have been compared with experimental ones observed in the case of MDA-MB-231 cells in the same operating conditions. RESULTS: The real and simulated distributions are consistent. In both cases the cells distribution near the electrodes is dominated by cell-cell dipole interactions which generate long chains. CONCLUSIONS: The agreement between real and simulated cells' distributions demonstrate the method's reliability. The distribution are dominated by cell-cell dipole interactions even at low density regimes (105 cell/ml). An improved estimate for the density threshold governing the interaction free regime is suggested.

Genetic, clinical and radiographic signs in knee osteoarthritis susceptibility.

INTRODUCTION: Osteoarthritis (OA) is considered to be a multifactorial and polygenic disease and diagnosis is mainly clinical and radiological. Correlation between radiographic data and clinical status has been reported. However, very few studies, especially in Caucasian people, describe the association between the Kellgren and Lawrence OA grading scale (KL) and genetic alterations to better understand OA etiopathogenesis and susceptibility. In order to update the knee OA grading, in this study we assessed the associations between KL grade, clinical features such as American Knee Society Score (AKSS), age, and polymorphisms in the principal osteoarthritis susceptibility (OS) genes in Sicilian individuals. METHODS: In 66 Sicilian individuals affected by primary knee OA, the clinical and radiographic evaluation was performed using 2 sub-scores of AKSS (knee score (KS) and function score (FS)) and KL. The patients were also classified according to age. Online Mendelian Inheritance in Man (OMIM) and Database of Single Nucleotide Polymorphisms (dbSNP) Short Genetic Variations databases were used to select gene regions containing the following polymorphisms to analyze: FRZB rs288326 and rs7775, MATN3 rs77245812, ASPN D14 repeats, PTHR2 rs76758470, GDF5 rs143383 and DVWA rs11718863. Patient genotypes were obtained using Sanger DNA sequencing analysis. RESULTS: In our cohort of patients a statistical association between the variables analyzed was reported in all associations tested (KL versus KS, FS and age). We observed that a mild to severe OA radiographic grade is related to severe clinical conditions and loss of articular function and that the severity of symptoms increases with age. Concerning the genotyping analysis, our results revealed a significant statistical association between KL grading and GDF5 rs143383 and DVWA rs11718863 genetic alterations. The latter was also associated with a more severe radiographic grade, displaying its predictive role as OA marker progression. Statistically significant association between clinical, radiographic and genetic signs observed, suggests extending the actual grading of knee OA based mainly on X-ray features. CONCLUSIONS: This work represents a multidisciplinary and translational medicine approach to study OA where clinical, radiological, and OS5 and OS6 SNPs evaluation could contribute to better define grading and progression of OA and to the development of new therapies.

Genetics of longevity. data from the studies on Sicilian centenarians.

The demographic and social changes of the past decades have determined improvements in public health and longevity. So, the number of centenarians is increasing as a worldwide phenomenon. Scientists have focused their attention on centenarians as optimal model to address the biological mechanisms of "successful and unsuccessful ageing". They are equipped to reach the extreme limits of human life span and, most importantly, to show relatively good health, being able to perform their routine daily life and to escape fatal age-related diseases, such as cardiovascular diseases and cancer. Thus, particular attention has been centered on their genetic background and immune system. In this review, we report our data gathered for over 10 years in Sicilian centenarians. Based on results obtained, we suggest longevity as the result of an optimal performance of immune system and an over-expression of anti-inflammatory sequence variants of immune/inflammatory genes. However, as well known, genetic, epigenetic, stochastic and environmental factors seem to have a crucial role in ageing and longevity. Epigenetics is associated with ageing, as demonstrated in many studies. In particular, ageing is associated with a global loss of methylation state. Thus, the aim of future studies will be to analyze the weight of epigenetic changes in ageing and longevity.

Rapid KRAS, EGFR, BRAF and PIK3CA mutation analysis of fine needle aspirates from non-small-cell lung cancer using allele-specific qPCR.

Endobronchial Ultrasound Guided Transbronchial Needle Aspiration (EBUS-TBNA) and Trans-esophageal Ultrasound Scanning with Fine Needle Aspiration (EUS-FNA) are important, novel techniques for the diagnosis and staging of non-small cell lung cancer (NSCLC) that have been incorporated into lung cancer staging guidelines. To guide and optimize treatment decisions, especially for NSCLC patients in stage III and IV, EGFR and KRAS mutation status is often required. The concordance rate of the mutation analysis between these cytological aspirates and histological samples obtained by surgical staging is unknown. Therefore, we studied the extent to which allele-specific quantitative real-time PCR with hydrolysis probes could be reliably performed on EBUS and EUS fine needle aspirates by comparing the results with histological material from the same patient. We analyzed a series of 43 NSCLC patients for whom cytological and histological material was available. We demonstrated that these standard molecular techniques can be accurately applied on fine needle cytological aspirates from NSCLC patients. Importantly, we show that all mutations detected in the histological material of primary tumor were also identified in the cytological samples. We conclude that molecular profiling can be reliably performed on fine needle cytology aspirates from NSCLC patients.

Mung bean nuclease mapping of RNAs 3' end.

A method is described that allows an accurate mapping of 3' ends of RNAs. In this method a labeled DNA probe, containing the presumed 3' end of the RNA under analysis is allowed to anneals to the RNA itself. Mung-bean nuclease is then used to digest single strands of both RNA and DNA. Electrophoretic fractionation of "protected" undigested, labeled DNA is than performed using a sequence reaction of a known DNA as length marker. This procedure was applied to the analysis of both a polyA RNA (Interleukin 10 mRNA) and non polyA RNAs (sea urchin 18S and 26S rRNAs). This method might be potentially relevant for the evaluation of the role of posttrascriptional control of IL-10 in the pathogenesis of the immune and inflammatory mediated diseases associated to ageing. This might allow to develop new strategies to approach to the diagnosis and therapy of age related diseases.

Analysis of HLA-DRB1, DQA1, DQB1 haplotypes in Sardinian centenarians.

Some genetic determinants of longevity might reside in those polymorphisms for the immune system genes that regulate immune responses. Many longevity association studies focused their attention on HLA (the human MHC) polymorphisms, but discordant results have been obtained. Sardinians are a relatively isolate population and represent a suitable population for association studies. Some HLA-DR and DQ alleles form very stable haplotypes with a strong linkage disequilibrium. In a previous study on Sardinian centenarians we have suggested that HLA-DRB1 *15 allele might be marginally associated to longevity. HLA-DR,DQ haplotypes are in strong linkage disequilibrium and well conserved playing a role in the association to diseases. Hence, we have evaluated, by amplification refractory mutation system/polymerase chain reaction (ARMS-PCR) the HLADQA1 and HLA-DQB1 allele frequencies in 123 centenarians and 92 controls from Sardinia to assess whether the association to HLA-DRB1 *15 allele may be due to the other genes involved in the HLA-DR,DQ haplotypes. The frequencies of HLA-DQA1, DQB1 haplotypes were not significantly modified in centenarians. Nevertheless by evaluating the frequency of DRB1 *15 linked haplotypes, we observed a not significant increase in centenarians of HLA-DQA1 *01, DQB1 *05 and HLA-DQA1 *01,DQB1 *06 haplotypes. These data suggest that these haplotypes might have a role in determining life span expectancy and longevity.

Apolipoprotein E genotypic frequencies among Down syndrome patients imply early unsuccessful aging for ApoE4 carriers.

Down syndrome (DS) might be considered a model for unsuccessful and early aging, possibly accelerated for those who carry the APOE4 allele associated with common age-related diseases, e.g., Alzheimer's disease and a poor prognosis after acute myocardial infarction, causing lower ApoE4 frequencies among the very old in general populations. We compared ApoE genotypic frequencies found for healthy adults (n = 211, age < 40; n = 79, ages 70-79; n = 71, ages > 90) to those found for DS patients (n = 106, mean age 9 years), all living in western Sicily. We found that the frequency of the ApoE23 genotype increased with age among the healthy adults (8.5%, 6.4%, 19.7%; p = 0.024) while ApoE34 frequency decreased (16.1%, 12.6%, 4.1%; p = 0.012). DS patients had APOE34 genotypic frequencies very similar to those found in septuagenarians (9%; p = 0.005). Analyzing results according to surviving rate of persons with DS, an age-related reduction of ApoE3/4 genotype frequency was found comparing =5 years old to >5 years old DS subjects. These results highlight DS as a model to understand the role of APOE4 allele in unsuccessful ageing considering that a number of proinflammatory supernumerary genes (Cu/Zn superoxide dismutase, Ets-2 transcription factors, Down syndrome critical region 1, stress-inducible factor, interferon-alpha receptor and the amyloid precursor protein) are located on chromosome 21 and are implied in the pathologic processes of DS.

Study of the association with -330T/G IL-2 in a population of centenarians from centre and south Italy.

Immune response in elderly is characterised by a progressive loss of the ability to cope environmental stressors with a characteristic remodelling of cytokine network. One of the data constantly reported in literature is the decrease of IL-2 production. An IL-2 central role in the reconstitution of T cell function in vitro is largely documented. Studies on a T-->G polymorphism at -330 nt of IL-2 gene promoter region have demonstrated that T lymphocytes from 330GG homozygous subjects are able to produce in vitro higher amount of IL-2, than -330TG heterozygous or -330TT homozygous subjects. As a genetic background conditioning the maintaining of an efficient immune response would exert positive effects on healthy ageing, we have typed for 330T/G IL-2 single nucleotide polymorphism (SNP), 168 centenarians and 214 control subjects matched for age and ancestry from Centre and South Italy to check the 330GG genotype association to longevity. The statistical analysis doesn't show a significant difference of genotypic and allelic frequencies at -330 IL-2 T/G SNP among centenarians and controls. Comparing the two cohorts of subjects by extended Mantel Haenszel procedure a marginally significant trend for an increased -330TT genotype frequency was observed. Our data seem to be paradoxical considering the role attributed to a well-conserved T cell function in the successful ageing. On the other hand, in a recent study on a sample of Irish octogenarians a similar distribution of -330T/G genotype even was observed. These data suggest that a genetic background favouring an increased IL-2 production might be detrimental for longevity. On the other hand, an increase of IL-2 and other pro-inflammatory cytokine production characterise the Alzheimer's disease serum profile. All in all our data seem to suggest that reduction of -330G allele frequency might be protective for healthy ageing limiting cell mediated inflammation implied in age associated diseases.