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.

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.

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.

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.

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.

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.

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.

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.

Evidence of 18F-FCH Uptake in Human T98G Glioblastoma Cells.

AIM: Tumor and chemo/radiotherapy-damaged brain tissues are hardly distinguishable by conventional morphological imaging. (18)F-FCH was compared against (18)F-FDG in the T98G glioblastoma cell line with regard to their radiopharmaceutical uptake, in order to test its diagnostic power on brain tumor lesions. MATERIALS AND METHODS: Equimolar amounts of (18)F-FCH and (18)F-FDG were added to human glioblastoma T98G cells and human dermal fibroblasts for 20, 40, 60, 90 and 120 min of incubation. Radiopharmaceutical uptake was expressed as a percentage of the administered dose. Cold choline was used for binding competition experiments. RESULTS: In T98G cells (18)F-FCH was taken-up in higher amounts than 18F-FDG after 60 min. In fibroblasts, uptake was lower than 1% for both radiopharmaceuticals. Cold choline reduced the uptake of FCH to 1% similarly to fibroblasts. CONCLUSION: Our results prove the efficacy of (18)F-FCH as a promising tracer, better than (18)F-FDG in establishing the tumor-to-background ratio in brain tumors.

Gallic acid exerts a protective or an anti-proliferative effect on glioma T98G cells via dose-dependent epigenetic regulation mediated by miRNAs.

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor in adulthood, characterized by very high recurrence. Following the limited results for conventional therapies, novel therapeutic agents are under investigation. Among the putative new molecules, gallic acid (GA) represents a promising new anticancer drug. The anticancer effect of this drug has been based on its antioxidant effects. The aim of the present study was to investigate the toxic effects of GA on the T98G human glioblastoma cell line and its capacity to modulate the expression of microRNAs targeting the genes involved in tumor growth and invasion. Cytotoxicity, clonogenic ability and cell migration after GA treatment were tested. Moreover, the expression of miRNAs that target genes for antioxidant mitochondrial enzymes (mir-17-3p), p-21 protein (mir-21-5p) and ATM (mir-421-5p) was determined by qRT-PCR. The results confirmed in the T98G cells the anti-proliferative effect of GA reported for other glioma cell lines and showed that the miRNA expression changes depending on GA concentrations. Different GA concentrations can determine a protective or a toxic effect on tumor cells. Thus, the key for GA to induce a specific anticancer action is to use an optimal concentration that avoids these twin effects.

Effects of single or combined treatments with radiation and chemotherapy on survival and danger signals expression in glioblastoma cell lines.

The success of chemo- and radiotherapy in glioblastoma multiforme, the most common and lethal primary brain tumour, could rely on the induction of immunogenic tumour cell death and on the induction of anticancer immune response. In this study we investigated cell survival to single treatments or combination of X-rays and temozolomide in glioblastoma cell lines (T98G and U251MG) and we attempted to identify danger signals (HMGB1 and HSP70) released by dying cells in the microenvironment that could activate antitumour immunity contributing to the therapeutic efficacy of conventional treatments. Our data suggest that HSP70 translocates from cytoplasm to extracellular environment after an increase in radiation dose and HMGB1 translocates from the nucleus to the cytoplasm and subsequently is released into the extracellular space, confirming a role of these proteins as signals released after radiation-induced damage in glioblastoma cells. We also could state that TMZ had limited effectiveness in activating HMGB1 and HSP70 signalling and, instead, an adjuvant effect was observed in some combined treatments, depending on schedule, cell line, and timing. A big challenge in tumour therapy is, therefore, to identify the most beneficial combination and chronology of multiple treatment options to contribute to the improvement of the therapeutic outcome.

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.

Late oral mucosa alterations after radiotherapy for head and neck cancer assessed by exfoliative cytology.

AIM: Late oral mucosa changes after radiotherapy for head and neck cancer have been poorly studied. This study aimed to determine long-term effects of radiotherapy on oral mucosa using exfoliative oral cytology. PATIENTS AND METHODS: Fifty patients with cancer were enrolled, five of whom in order to validate microscopic analysis. Smears were collected at programmed visit; a score was used to rank possible cytological alterations. Presence of inflammation was also microscopically described and compared to blood count tests. RESULTS: Epithelial cells revealed a peculiar 'folding' phenotype, not related to chemotherapy, total dose, or to the effective dose delivered to mucosa. Inflammation described was related to the score for 'folding' cells; moreover, score decreased in the presence of a higher lymphocyte count, while it was not altered by neutrophil count. CONCLUSION: We suggest application of exfoliative cytology to study radiation injury and the variability of individual response of oral mucosa to radiation.

New frontiers for astrocytic tumours.

Glioblastoma multiforme, the most common type of primary brain tumour, remains an unsolved clinical problem. A great deal of work has been done in an effort to understand the biology and genetics of glioblastoma multiforme, but clinically effective treatments remain elusive. It is well known that malignant gliomas develop resistance to chemo- and radiotherapy. In this review we evaluated the literature data regarding therapeutic progress for the treatment of astrocytic tumours, focusing our attention on new frontiers for glioblastoma. The research studies performed in in vitro and in vivo models show that the application of hyperthermia using magnetic nanoparticles is safe and could be a promising tool in the treatment of glioblastoma patients. Our efforts are focused towards new fields of research, for example nanomedicine and the study of the uptake and cytotoxic effects of magnetic nanoparticles. The improvement of the quality of life of patients, by increasing their survival rate is the best result to be pursued, since these tumours are considered as ineradicable.

Cell death forms and HSP70 expression in U87 cells after ionizing radiation and/or chemotherapy.

BACKGROUND: Several types of cell death can induce the activation of the immune system by releasing factors of damage. AIM: To identify different cell death modalities using U87 glioblastoma cell line after radio-chemotherapy treatments by analyzing the expression of HSP70 after γ-ray irradiation and temozolomide treatment. MATERIALS AND METHODS: U87 cell line was irradiated with 2, 4, 8, 10 and 20 Gy, treated with 200 µM of temozolomide, or subjected to combined treatments of these. RESULTS: Forty-eight hours after treatment, apoptosis was more prevalent than necrosis, especially after γ-radiation with a somewhat dose-dependent trend. Temozolomide did not seem to induce significant modifications of the pattern of cell death and protein expression. Temozolomide-induced cell death was lower or equal to that obtained with high dose radiotherapy. A low synergism between the two treatments was observed. CONCLUSION: This cell line showed a marked radioresistance to conventional clinical doses, showing attempts to repair the induced damage, while temozolomide treatment showed no cytotoxic effects.

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.