A Phyto-mycotherapeutic Supplement, Namely Ganostile, as Effective Adjuvant in Brain Cancer Management: An In Vitro Study Using U251 Human Glioblastoma Cell Line.

The current standard oncotherapy for glioblastoma is limited by several adverse side effects, leading to a short-term patient survival rate paralleled by a worsening quality of life (QoL). Recently, Complementary and Integrative Medicine's (CIM) innovative approaches have shown positive impacts in terms of better response to treatment, side effect reduction, and QoL improvement. In particular, promising potential in cancer therapy has been found in compounds coming from phyto- and mycotherapy. The objective of this study was to demonstrate the beneficial effects of a new phyto-mycotherapy supplement, named Ganostile, in the human glioblastoma cell line U251, in combination with chemotherapeutic agents, i.e., Cisplatin and a new platinum-based prodrug. Choosing a supplement dosage that mimicked oral supplementation in humans (about 1 g/day), through in vitro assays, microscopy, and cytometric analysis, it has emerged that the cells, after 48hr continuous exposure to Ganostile in combination with the chemical compounds, showed a higher mortality and a lower proliferation rate than the samples subjected to the different treatments administered individually. In conclusion, our data support the use of Ganostile in integrative oncology protocols as a promising adjuvant able to amplify conventional and new drug effects and also reducing resistance mechanisms often observed in brain tumors.

Modifications of Blood Molecular Components after Treatment with Low Ozone Concentrations.

The ex vivo treatment of a limited volume of blood with gaseous oxygen-ozone (O2-O3) mixtures and its rapid reinfusion into the patient is a widespread medical procedure. O3 instantly reacts with the blood's antioxidant systems, disappearing before reinfusion, although the molecules formed act as messengers in the organism, inducing multiple antioxidant and anti-inflammatory responses. An appropriate dose of O3 is obviously essential to ensure both safety and therapeutic efficacy, and in recent years, the low-dose O3 concept has led to a significant reduction in the administered O3 concentrations. However, the molecular events triggered by such low concentrations in the blood still need to be fully elucidated. In this basic study, we analysed the molecular modifications induced ex vivo in sheep blood by 5 and 10 µg O3/mL O2 by means of a powerful metabolomics analysis in association with haemogas, light microscopy and bioanalytical assays. This combined approach revealed increased oxygenation and an increased antioxidant capacity in the O3-treated blood, which accorded with the literature. Moreover, original information was obtained on the impact of these low O3 concentrations on the metabolic pathways of amino acids, carbohydrates, lipids and nucleotides, with the modified metabolites being mostly involved in the preservation of the oxidant-antioxidant balance and in energy production.

Ozone and procaine increase secretion of platelet-derived factors in platelet-rich plasma.

Platelet-rich plasma (PRP) is gaining more and more attention in regenerative medicine as an innovative and efficient therapeutic approach. The regenerative properties of PRP rely on the numerous bioactive molecules released by the platelets: growth factors are involved in proliferation and differentiation of endothelial cells and fibroblasts, angiogenesis and extracellular matrix formation, while cytokines are mainly involved in immune cell recruitment and inflammation modulation. Attempts are ongoing to improve the therapeutic potential of PRP by combining it with agents able to promote regenerative processes. Two interesting candidates are ozone, administered at low doses as gaseous oxygen-ozone mixtures, and procaine. In the present study, we investigated the effects induced on platelets by the in vitro treatment of PRP with ozone or procaine, or both. We combined transmission electron microscopy to obtain information on platelet modifications and bioanalytical assays to quantify the secreted factors. The results demonstrate that, although platelets were already activated by the procedure to prepare PRP, both ozone and procaine induced differential morpho-functional modifications in platelets resulting in an increased release of factors. In detail, ozone induced an increase in surface protrusions and open canalicular system dilation suggestive of a marked α-granule release, while procaine caused a decrease in surface protrusions and open canalicular system dilation but a remarkable increase in microvesicle release suggestive of high secretory activity. Consistently, nine of the thirteen platelet-derived factors analysed in the PRP serum significantly increased after treatment with ozone and/or procaine. Therefore, ozone and procaine proved to have a remarkable stimulating potential without causing any damage to platelets, probably because they act through physiological, although different, secretory pathways.

Physical Training Chronically Stimulates the Motor Neuron Cell Nucleus in the Ts65Dn Mouse, a Model of Down Syndrome.

Down syndrome (DS) is a genetically-based disease based on the trisomy of chromosome 21 (Hsa21). DS is characterized by intellectual disability in association with several pathological traits among which early aging and altered motor coordination are prominent. Physical training or passive exercise were found to be useful in counteracting motor impairment in DS subjects. In this study we used the Ts65Dn mouse, a widely accepted animal model of DS, to investigate the ultrastructural architecture of the medullary motor neuron cell nucleus taken as marker of the cell functional state. Using transmission electron microscopy, ultrastructural morphometry, and immunocytochemistry we carried out a detailed investigation of possible trisomy-related alteration(s) of nuclear constituents, which are known to vary their amount and distribution as a function of nuclear activity, as well as the effect of adapted physical training upon them. Results demonstrated that trisomy per se affects nuclear constituents to a limited extent; however, adapted physical training is able to chronically stimulate pre-mRNA transcription and processing activity in motor neuron nuclei of trisomic mice, although to a lesser extent than in their euploid mates. These findings are a step towards understanding the mechanisms underlying the positive effect of physical activity in DS.

Mitochondrial Features of Mouse Myoblasts Are Finely Tuned by Low Doses of Ozone: The Evidence In Vitro.

The mild oxidative stress induced by low doses of gaseous ozone (O3) activates the antioxidant cell response through the nuclear factor erythroid 2-related factor 2 (Nrf2), thus inducing beneficial effects without cell damage. Mitochondria are sensitive to mild oxidative stress and represent a susceptible O3 target. In this in vitro study, we investigated the mitochondrial response to low O3 doses in the immortalized, non-tumoral muscle C2C12 cells; a multimodal approach including fluorescence microscopy, transmission electron microscopy and biochemistry was used. Results demonstrated that mitochondrial features are finely tuned by low O3 doses. The O3 concentration of 10 µg maintained normal levels of mitochondria-associated Nrf2, promoted the mitochondrial increase of size and cristae extension, reduced cellular reactive oxygen species (ROS) and prevented cell death. Conversely, in 20 µg O3-treated cells, where the association of Nrf2 with the mitochondria drastically dropped, mitochondria underwent more significant swelling, and ROS and cell death increased. This study, therefore, adds original evidence for the involvement of Nrf2 in the dose-dependent response to low O3 concentrations not only as an Antioxidant Response Elements (ARE) gene activator but also as a regulatory/protective factor of mitochondrial function.

Ozone at low concentration modulates microglial activity in vitro: A multimodal microscopy and biomolecular study.

Oxygen-ozone (O2 -O3 ) therapy is an adjuvant/complementary treatment based on the activation of antioxidant and cytoprotective pathways driven by the nuclear factor erythroid 2-related factor 2 (Nrf2). Many drugs, including dimethyl fumarate (DMF), that are used to reduce inflammation in oxidative-stress-related neurodegenerative diseases, act through the Nrf2-pathway. The scope of the present investigation was to get a deeper insight into the mechanisms responsible for the beneficial result of O2 -O3 treatment in some neurodegenerative diseases. To do this, we used an integrated approach of multimodal microscopy (bright-field and fluorescence microscopy, transmission and scanning electron microscopy) and biomolecular techniques to investigate the effects of the low O3 concentrations currently used in clinical practice in lipopolysaccharide (LPS)-activated microglial cells human microglial clone 3 (HMC3) and in DMF-treated LPS-activated (LPS + DMF) HMC3 cells. The results at light and electron microscopy showed that LPS-activation induced morphological modifications of HMC3 cells from elongated/branched to larger roundish shape, cytoplasmic accumulation of lipid droplets, decreased electron density of the cytoplasm and mitochondria, decreased amount of Nrf2 and increased migration rate, while biomolecular data demonstrated that Heme oxygenase 1 gene expression and the secretion of the pro-inflammatory cytokines, Interleukin-6, and tumor necrosis factor-α augmented. O3 treatment did not affect cell viability, proliferation, and morphological features of both LPS-activated and LPS + DMF cells, whereas the cell motility and the secretion of pro-inflammatory cytokines were significantly decreased. This evidence suggests that modulation of microglia activity may contribute to the beneficial effects of the O2 -O3 therapy in patients with neurodegenerative disorders characterized by chronic inflammation. HIGHLIGHTS: Low-dose ozone (O3 ) does not damage activated microglial cells in vitro Low-dose O3 decreases cell motility and pro-inflammatory cytokine secretion in activated microglial cells in vitro Low-dose O3 potentiates the effect of an anti-inflammatory drug on activated microglial cells.