Radiation-induced circulating miRNA expression in blood of head and neck cancer patients.

In recent years, scientists have found evidence confirming the aberrant expression of miRNAs in cancer patients compared to healthy individuals. The growing interest in the identification of non-invasive and specific diagnostic and prognostic molecular markers has identified microRNAs as potential candidates in cancer diagnosis, prognosis and treatment response. In the present study, we have analyzed the expression profile of circulating miR-21, -191 and -421 in peripheral blood of head and neck cancer patients (HNC) to investigate a possible modulation of mRNA levels by radiation and to identify the role of mRNA as biomarkers of cancer prognosis. Results showed a modulation of the microRNA expression at different time points after radiotherapy, suggesting that treatment may influence the release of circulating miRNAs depending also on the time interval elapsed since radiotherapy. The expression levels of miR-21, -191 and -421 were higher in blood of patients treated with radiotherapy alone after 6 months from the end of therapy and high levels of them seemed to correlate with the remission of the disease. The trends shown in this study confirmed that miRNAs could be useful prognosis markers and could provide preliminary data for further evaluation in predicting patients' response to radiotherapy by developing miRNA-based treatments to improve the sensitivity of cancer cells to radiotherapy.

Rhamnose-coated superparamagnetic iron-oxide nanoparticles: an evaluation of their in vitro cytotoxicity, genotoxicity and carcinogenicity.

Tumor recurrence after the incomplete removal of a tumor mass inside brain tissue is the main reason that scientists are working to identify new strategies in brain oncologic therapy. In particular, in the treatment of the most malignant astrocytic tumor glioblastoma, the use of magnetic nanoparticles seems to be one of the most promising keys in overcoming this problem, namely by means of magnetic fluid hyperthermia (MFH) treatment. However, the major unknown issue related to the use of nanoparticles is their toxicological behavior when they are in contact with biological tissues. In the present study, we investigated the interaction of glioblastoma and other tumor cell lines with superparamagnetic iron-oxide nanoparticles covalently coated with a rhamnose derivative, using proper cytotoxic assays. In the present study, we focused our attention on different strategies of toxicity evaluation comparing different cytotoxicological approaches in order to identify the biological damages induced by the nanoparticles. The data show an intensive internalization process of rhamnose-coated iron oxide nanoparticles by the cells, suggesting that rhamnose moiety is a promising biocompatible coating in favoring cells' uptake. With regards to cytotoxicity, a 35% cell death at a maximum concentration, mainly as a result of mitochondrial damages, was found. This cytotoxic behavior, along with the high uptake ability, could facilitate the use of these rhamnose-coated iron-oxide nanoparticles for future MFH therapeutic treatments.

Effects of extremely low-frequency magnetotherapy on proliferation of human dermal fibroblasts.

Extremely low-frequency electromagnetic fields (ELF-EMFs) applied in magnetotherapy have frequency lower than 100 Hz and magnetic field intensity ranging from 0.1 to 20 mT. For many years, the use of magnetotherapy in clinics has been increasing because of its beneficial effects in many processes, e.g., skin diseases, inflammation and bone disorders. However, the understanding of the microscopic mechanisms governing such processes is still lacking and the results of the studies on the effects of ELF-EMFs are controversial because effects derive from different conditions and from intrinsic responsiveness of different cell types.In the present study, we studied the biological effects of 1.5 h exposure of human dermal fibroblasts to EMFs with frequencies of 5 and 50 Hz and intensity between 0.25 and 1.6 mT. Our data showed that the magnetic treatment did not produce changes in cell viability, but gave evidence of a sizeable decrease in proliferation at 24 h after treatment. In addition, immunofluorescence experiments displayed an increase in tubulin expression that could foreshadow changes in cell motility or morphology. The decrease in proliferation with unchanged viability and increase in tubulin expression could be consistent with the triggering of a transdifferentiation process after the exposure to ELF-EMFs.

(18)F-FET and (18)F-FCH uptake in human glioblastoma T98G cell lines.

BACKGROUND: Despite complex treatment of surgery, radiotherapy and chemotherapy, high grade gliomas often recur. Differentiation between post-treatment changes and recurrence is difficult. (18)F-methyl-choline ((18)F-FCH) is frequently used in staging and detection of recurrent prostate cancer disease as well as some brain tumours; however accumulation in inflammatory tissue limits its specificity. The (18)F-ethyl-tyrosine ((18)F-FET) shows a specific uptake in malignant cells, resulting from increased expression of amino acid transporters or diffusing through the disrupted blood-brain barrier. (18)F-FET exhibits lower uptake in machrophages and other inflammatory cells. Aim of this study was to evaluate (18)F-FCH and (18)F-FET uptake by human glioblastoma T98G cells. MATERIAL AND METHODS: Human glioblastoma T98G or human dermal fibroblasts cells, seeded at a density to obtain 2 × 10(5) cells per flask when radioactive tracers were administered, grew adherent to the plastic surface at 37°C in 5% CO2 in complete medium. Equimolar amounts of radiopharmaceuticals were added to cells for different incubation times (20 to 120 minutes) for (18)F-FCH and (18)F-FET respectively. The cellular radiotracer uptake was determined with a gamma counter. All experiments were carried out in duplicate and repeated three times. The uptake measurements are expressed as the percentage of the administered dose of tracer per 2 × 10(5) cells. Data (expressed as mean values of % uptake of radiopharmaceuticals) were compared using parametric or non-parametric tests as appropriate. Differences were regarded as statistically significant when p<0.05. RESULTS: A significant uptake of (18)F-FCH was seen in T98G cells at 60, 90 and 120 minutes. The percentage uptake of (18)F-FET in comparison to (18)F-FCH was lower by a factor of more than 3, with different kinetic curves.(18)F-FET showed a more rapid initial uptake up to 40 minutes and (18)F-FCH showed a progressive rise reaching a maximum after 90 minutes. CONCLUSIONS: (18)F-FCH and (18)F-FET are candidates for neuro-oncological PET imaging. (18)F-FET could be the most useful oncological PET marker in the presence of reparative changes after therapy, where the higher affinity of (18)F-FCH to inflammatory cells makes it more difficult to discriminate between tumour persistence and non-neoplastic changes. Additional studies on the influence of inflammatory tissue and radionecrotic cellular components on radiopharmaceutical uptake are necessary.

NMR as evaluation strategy for cellular uptake of nanoparticles.

Advanced nanostructured materials, such as gold nanoparticles, magnetic nanoparticles, and multifunctional materials, are nowadays used in many state-of-the-art biomedical application. However, although the engineering in this field is very advanced, there remain some fundamental problems involving the interaction mechanisms between nanostructures and cells or tissues. Here we show the potential of (1)H NMR in the investigation of the uptake of two different kinds of nanostructures, that is, maghemite and gold nanoparticles, and of a chemotherapy drug (Temozolomide) in glioblastoma tumor cells. The proposed experimental protocol provides a new way to investigate the general problem of cellular uptake for a variety of biocompatible nanostructures and drugs.

Effects of Photons Irradiation on 18F-FET and 18F-DOPA Uptake by T98G Glioblastoma Cells.

The differential diagnosis between brain tumors recurrence and early neuroinflammation or late radionecrosis is still an unsolved problem. The new emerging magnetic resonance imaging, computed tomography, and positron emission tomography diagnostic modalities still lack sufficient accuracy. In the last years, a great effort has been made to develop radiotracers able to detect specific altered metabolic pathways or tumor receptor markers. Our research project aims to evaluate irradiation effects on radiopharmaceutical uptake and compare the kinetic of the fluorinate tracers. T98G glioblastoma cells were irradiated at doses of 2, 10, and 20 Gy with photons, and 18F-DOPA and 18F-FET tracer uptake was evaluated. Activity and cell viability at different incubation times were measured. 18F-FET and 18F-DOPA are accumulated via the LAT-1 transporter, but 18F-DOPA is further incorporated, whereas 18F-FET is not metabolized. Therefore, time-activity curves (TACs) tend to plateau with 18F-DOPA and to a rapid washout with 18F-FET. After irradiation, 18F-DOPA TAC resembles the 18F-FET pattern. 18F-DOPA activity peak we observed at 20 min might be fictitious, because earlier time points have not been evaluated, and a higher activity peak before 20 min cannot be excluded. In addition, the activity retained in the irradiated cells remains higher in comparison to the sham ones at all time points investigated. This aspect is similar in the 18F-FET TAC but less evident. Therefore, we can hypothesize the presence of a second intracellular compartment in addition to the amino acidic pool one governed by LAT-1, which could explain the progressive accumulation of 18F-DOPA in unirradiated cells.

Experimental and theoretical analysis of cytokine release for the study of radiation-induced bystander effect.

PURPOSE: To clarify the experimental conditions that might influence the release of cytokines in the culture medium and give some basic input for building a model for cytokine (e.g., Interleukin-6, IL-6) regulation in the case of 'sham irradiation' and after ionising radiation exposure. MATERIALS AND METHODS: The influence of cell type, cell density, medium volume, medium storage temperature and other methodological aspects on IL-6 and Interleukin-8 (IL-8) release were investigated. In addition, the effects over the time of different doses of gamma irradiation on the clonogenic survival of bystander cells and on the secretion of these cytokines were studied. RESULTS: We observed significant decreases of clonogenic survival in AG01522 and T98G cells after the transfer of medium collected 5 and 20 h after low doses of gamma irradiation. Concerning the Interleukins' measurements, our experiments showed that the aggregate removal modalities tested, and up to 10 freeze-thaw cycles, do not have significant influence on the measurements of IL-6 concentration in the medium. We also observed that the IL-6 accumulated in the medium of human fibroblasts is not degraded when maintained at 37 degrees C. Sets of experiments demonstrated that cell density or medium volume do not influence the release of IL-6. On the contrary, our results showed that IL-8 released by glioblastoma cells strongly depends on the amount of medium. Finally, the exposure of fibroblasts to gamma irradiation has influence on the release kinetics of both IL-6 and IL-8 with peculiar features. CONCLUSIONS: This study solved some of the methodological doubts concerning the study of bystander effects by means of the medium transfer technique; moreover it also highlighted some experimental aspects that need to be considered when approaching this sort of experiments.

The effects of alginate encapsulation on NIT-1 insulinoma cells: viability, growth and insulin secretion.

Transplantation of microencapsulated insulin-secreting cells is proposed as a promising therapy for the treatment of type I diabetes mellitus. In recent years, important advances have been made in the field of immunoisolation and many studies have shown that alginate provides some major advantages for encapsulation over other systems. Since it is known that the extracellular matrix influences the behaviour of encapsulated cells, the aim of the present work has been to study the consequences of encapsulation on some cell functions. For this purpose, cell growth and dynamics of insulin release of NIT-1 cells entrapped in alginate capsules compared with those exhibited by free NIT-1 cells were investigated by means of growth curves, assays, Trypan blue staining and ELISA test. All investigations performed allowed us to conclude that alginate-entrapped NIT-1 cells maintain their growth features and secretory functions although with some important differences. In particular, alginate encapsulation affects the cellular growth profile and causes the lost of time dependence of insulin secretion profile.

Effects of L-DOPA Pretreatment on the Kinetics, Migration and Carbon Ion Radiation Response of T98G Cells.

BACKGROUND/AIM: Glioblastoma is the most malignant and widespread brain tumor in adults, with a rapid clinical course. Recently, it has been hypothesized that L-DOPA plays a role in the diagnosis and treatment of glioblastoma. The aim of this study was to assess the effects of pretreatment with L-DOPA on the biological behavior of human T98G cells in vitro. MATERIALS AND METHODS: T98G cells were treated with 50 µg/ml or 100 µg/ml of L-DOPA for 4 h and their morphology, growth rate, clonogenic survival and migratory capacity in basal conditions and after carbon ion irradiation were evaluated using standard methods. RESULTS: Treated cells showed a lower growth rate and an increased migratory capacity that correlated with the dose of tested L-DOPA. Treatment with L-DOPA increased the growth rate of carbon ion irradiated T98G cells compared to control non-treated cells exposed to the same radiation dose. CONCLUSION: Our results open further questions about the overall advantage of L-DOPA treatment of glioblastoma.

Morphological Analysis of Amoeboid-Mesenchymal Transition Plasticity After Low and High LET Radiation on Migrating and Invading Pancreatic Cancer Cells.

BACKGROUND/AIM: Cell migration and invasion are fundamental components of tumor cell metastasis that represent the biggest threat to the survival and quality of life of cancer patients. There is clear evidence that ionizing radiation can differently modulate migration and invasiveness of cancer cells depending on the cell lines, the doses and the radiation types investigated. This suggests that motile cells are able to adopt different migration strategies according to their molecular characteristics and external signals. MATERIALS AND METHODS: In this study, a morphological analysis was performed on pancreatic cancer Aspc-1 cells to evaluate the amoeboid-mesenchymal mobility transition in several experimental conditions considering the role played by factors released by normal and tumor cells, in basal conditions and after low and high Linear Energy Transfer (LET) irradiation. RESULTS AND CONCLUSION: The migratory behavior of Aspc-1 cells is modulated by factors released by normal fibroblasts and tumor cells, and this is in turn modulated by both the radiation dose and the radiation quality.

Alternative splicing of hTERT: a further mechanism for the control of active hTERT in acute myeloid leukemia.

hTERT component is the key regulator of telomerase. Alternatively spliced variants of hTERT generate different telomerase activity. The goal of the study was to determine the role of different hTERT isoforms in the regulation of telomerase expression in AML patients. Among the 97 studied patients, 45 had a complex karyotype and 52 a normal karyotype. hTERT isoforms expression was determined in bone marrow samples by q-RT-PCR, using SYBR Green I. hTERT expression was lower in AML patients than controls (median 2.5 vs. 10.1, p = .003), though no difference was observed between the complex and normal karyotype (median 3.2 vs. 2.3, p = .37). High trans-dominant negative isoform expression increased the response rate by two. High expression of inactive product (-α - ß) was shown to increase the risk of relapse by about three times. In conclusion, our data suggest an intriguing link between the control of hTERT isoforms expression and AML outcome.

Matrix metalloprotease activity is enhanced in the compensated but not in the decompensated phase of pressure overload hypertrophy.

BACKGROUND: During the transition of pressure overload hypertrophy (POH) to heart failure (HF) there is intense interstitial cardiac remodeling, characterized by a complex balance between collagen deposition and degradation by matrix metalloproteases (MMPs). This study was aimed at investigating the process of cardiac remodeling during the different phases of the transition of POH to HF. METHODS: Guinea pigs underwent thoracic descending aortic banding or sham operation. Twelve weeks after surgery, left-ventricular (LV) end-diastolic internal dimension and ventricular systolic pressure were measured by combined M-mode echocardiography and micromanometer cathetherization. The MMP activity, tissue-specific MMP inhibitors (TIMPs), and collagen fraction were evaluated in LV tissue samples by zymography, ELISA, and computer-aided analysis, respectively. RESULTS: Banded animals were divided by lung weight values into either compensated left-ventricular hypertrophy (LVH) or HF groups, as compared with sham-operated controls. All HF animals exhibited a restrictive pattern of Doppler transmitral inflow, indicative of diastolic dysfunction, and developed lung congestion. Compensated LVH was associated with increased MMP-2 activity, which was blunted after transition to HF, at a time when TIMP-2 levels and collagen deposition were increased. CONCLUSIONS: The cardiac remodeling process that accompanies the development of POH is a phase-dependent process associated with progressive deterioration of cardiac function.

MDS/AML del(11)(q14) Share Common Morphological Features Despite Different Chromosomal Breakpoints.

In myelodysplatic syndromes and acute myeloid leukemia (MDS/AML) deletion of the 11q14 region is a rare chromosomal defect (incidence: 0.6-1.0%), included within the intermediate risk criteria by the International Prognostic Scoring System. No fluorescence in situ hybridization (FISH) study has yet been performed to identify a common breakpoint region (CBR). In our study through FISH with bacterial artificial chromosomes and commercial probes, we analyzed seven patients with MDS/AML harboring 11q14 deletion on conventional cytogenetic analysis. FISH revealed deletions in five patients and amplifications in two. Three patients with deletion carried a CBR, two had a deletion involving a more centromeric breakpoint. These five patients exhibited multilineage dysplasia, blast cells with large round nuclei, loose chromatin, small and abundant nucleoli, and vacuolated cytoplasm with very thin Auer bodies. In conclusion, the morphological features which occur independently of the extent of the deletion are of multilineage dysplasia in MDS and leukemic blasts strongly reactive to peroxidase in AML; despite the variable size of the deleted area, some patients harbor a CBR.

Uptake of 18F-FET and 18F-FCH in Human Glioblastoma T98G Cell Line after Irradiation with Photons or Carbon Ions.

The differential diagnosis between recurrence of gliomas or brain metastases and this phenomenon is important in order to choose the best therapy and predict the prognosis but is still a big problem for physicians. The new emerging MRI, CT, and PET diagnostic modalities still lack sufficient accuracy. Radiolabeled choline and amino acids have been reported to show great tumor specificity. We studied the uptake kinetics of [18F]fluoromethyl-choline (FCH) and O-(2-[18F]fluoroethyl)-L-tyrosine (FET) by the T98G human glioblastoma cells from 20 to 120 min after irradiation either with photons at 2-10-20 Gy or with carbon ions at 2 Gy (at the National Centre for Oncological Hadrontherapy (CNAO), Pavia, Italy). We also evaluated the cell death and morphology changes induced by radiation treatment. Both FET and FCH are able to trace tumor behavior in terms of higher uptake for increased doses of radiation treatment, due to the upregulation of cells attempts to repair nonlethal damage. Our data suggest that both FCH and FET could be useful to analyze the metabolic pathways of glioblastoma cells before and after radiotherapy. Physicians will have to consider the different kinetics pathways of uptake concerning the two radiopharmaceuticals.

Nuclear mRNA retention and aberrant doppel protein expression in human astrocytic tumor cells.

Doppel (Dpl) is a paralogue of the mammalian Prion (PrP) protein. It is abundant in testis and, unlike PrP, it is expressed at low levels in the adult central nervous system (CNS). Besides, Dpl overexpression correlates with some prion-disease pathological features, such as ataxia and death of cerebellar neurons. Recently, ectopic expression of doppel was found in two different tumor types, specifically in glial and haematological cancers. In this study the doppel gene (PRND) mRNA and protein expression in PRT-HU2 and IPDDC-A2 astrocytoma-derived cell lines was investigated. Northern blot analysis revealed two equally abundant PRND mRNA isoforms, while real-time PCR, on nuclear and cytoplasmic RNA fractions, and cRNA in situ hybridization, on astrocytoma cells and bioptical specimens, showed a nuclear retention of PRND transcripts. Western blot analysis showed that the amount of protein expressed is low compared to the level of mRNA. Moreover deglycosylation studies indicated that Dpl undergoes unusual glycosylation processes. Immunohistochemistry experiments demonstrated that Dpl was mainly localised in the cytoplasm of the astrocytic tumor cells, and that it failed to be GPI-anchored to the cell membrane. This unusual cellular localization was also confirmed through EGFP-Dpl expression in astrocytomas; on the contrary, HeLa cells exhibited the expected Dpl membrane localization. Our findings suggest an aberrant doppel gene expression pattern, characterized by a substantial nuclear retention of the transcript, an altered post-translational modification of the protein and an unusual cytoplasmic localization.

Human leukocyte antigen and antigen processing machinery component defects in astrocytic tumors.

PURPOSE: To determine the frequency of abnormalities in human leukocyte antigen (HLA) and antigen processing machinery (APM) component expression in malignant brain tumors. This information may contribute to our understanding of the immune escape mechanisms used by malignant brain tumors because HLA antigens mediate interactions of tumor cells with the host's immune system. EXPERIMENTAL DESIGN: Eighty-eight surgically removed malignant astrocytic tumors, classified according to the WHO criteria, were stained in immunoperoxidase reactions with monoclonal antibody recognizing monomorphic, locus-specific, and allospecific determinants of HLA class I antigens, beta2-microglobulin, APM components (LMP2, LMP7, TAP1, TAP2, calnexin, calreticulin, and tapasin), and HLA class II antigens. RESULTS: HLA class I antigens were lost in approximately 50% of the 47 glioblastoma multiforme (GBM) lesions and in approximately 20% of the 18 grade 2 astrocytoma lesions stained. Selective HLA-A2 antigen loss was observed in approximately 80% of the 24 GBM lesions and in approximately 50% of the 12 grade 2 astrocytoma lesions stained. HLA class I antigen loss was significantly (P < 0.025) correlated with tumor grade. Among the APM components investigated, tapasin expression was down-regulated in approximately 20% of the GBM lesions analyzed; it was associated, although not significantly, with HLA class I antigen down-regulation and tumor grade. HLA class II antigen expression was detected in approximately 30% of the 44 lesions analyzed. CONCLUSION: The presence of HLA antigen defects in malignant brain tumors may provide an explanation for the relatively poor clinical response rates observed in the majority of the T cell-based immunotherapy clinical trials conducted to date in patients with malignant brain tumors.

Radium-223: Insight and Perspectives in Bone-metastatic Castration-resistant Prostate Cancer.

223Ra prolongs overall survival in symptomatic patients affected by multiple bone-metastatic castration-resistant prostatic cancer, without visceral or nodal involvement. However, many questions remain about its mechanisms of action, and its use in clinical practice is still unresolved. First of all, what is the main target of alpha-particle emission, that is, in what way does it influences the tumor microenvironment? When is the best timing in the course of the disease, extending its use to asymptomatic low-volume or even to the micrometastatic phase? What are suitable biomarkers to be employed as prognostic factors and response indicators? Which associations with other drugs and their sequence can offer the best results, and is their effect additive or synergistic? Ultimately, in the current climate of spending review, what is the optimal cost and benefit ratio regarding available treatments? In this review, we tried to answer these questions by analyzing the available scientific literature.

Pulsed Electromagnetic Field with Temozolomide Can Elicit an Epigenetic Pro-apoptotic Effect on Glioblastoma T98G Cells.

Treatment with pulsed electromagnetic fields (PEMFs) is emerging as an interesting therapeutic option for patients with cancer. The literature has demonstrated that low-frequency/low-energy electromagnetic fields do not cause predictable effects on DNA; however, they can epigenetically act on gene expression. The aim of the present work was to study a possible epigenetic effect of a PEMF, mediated by miRNAs, on a human glioblastoma cell line (T98G). We tested a PEMF (maximum magnetic induction, 2 mT; frequency, 75 Hz) that has been demonstrated to induce autophagy in glioblastoma cells. In particular, we studied the effect of PEMF on the expression of genes involved in cancer progression and a promising synergistic effect with temozolomide, a frequently used drug to treat glioblastoma multiforme. We found that electromagnetic stimulation in combination with temozolomide can elicit an epigenetic pro-apoptotic effect in the chemo- and radioresistant T98G glioblastoma cell line.

Antibodies to cerebellar nerve fibres in two patients with paraneoplastic cerebellar ataxia.

The aim of this study was to characterize two new atypical anti-neuronal antibodies using an immunohistochemical method on rat cerebellum and Western blot techniques with primate cerebellar tissue and with recombinant neuronal proteins. Atypical sera from two patients with paraneoplastic neurological syndromes associated with different tumours were detected. Case number 1 presented cerebellar degeneration and Merkel cell carcinoma and case number 2 paraneoplastic brainstem encephalitis and malignant fibrous histiocytoma. By immunohistochemistry, the two new atypical antibodies showed a similar fibrillar positivity in the molecular and granular layers and around the Purkinje cells. The dot blot with recombinant neuronal proteins (HuD, NOVA-1, CDR62/Yo, Amphiphysin) was negative, whereas the Western blot with neuronal antigens of primate cerebellum identified two different proteins with molecular weights (64 kD in case number 1, and 70 kD in case number 2). In conclusion, the two new antibody reactivities against nerve fibres should be integrated into the diagnostic paraneoplastic neurological syndromes guidelines.

The immunobiology of malignant gliomas.

In spite of continuing advances in surgery, radiotherapy and chemotherapy, the last few decades have brought little improvement in the prognosis of malignant brain tumours. New immunotherapeutic strategies are being studied in numerous laboratories throughout the world in an effort to gather the further clinical and biological data needed in order to render these therapies effective and selective. The authors review recent advances in understanding of the immunobiology of brain tumours, in particular malignant gliomas. The biological basis of the interaction between nervous system cells and cancer cells is still an unresolved question and a constantly evolving area of research. Particular attention is paid to interleukin-2, one of the few effective cytokines used in the adjuvant treatment of some tumours. However, there is still a long way to go before these and other immunological approaches can be considered really applicable in the treatment of malignant gliomas.