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.

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.

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. 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. 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. 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 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.

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.