Daniele Maggioni

Apigenin impairs oral squamous cell carcinoma growth in vitro inducing cell cycle arrest and apoptosis

In the present study, we investigated the effect of apigenin, a flavonoid widely present in fruits and vegetables, on a tongue oral cancer-derived cell line (SCC-25) and on a keratinocyte cell line (HaCaT), with the aim of unveiling its antiproliferative mechanisms. The effect of apigenin on cell growth was evaluated by MTT assay, while apoptosis was investigated by phosphatidyl serine membrane translocation and cell cycle distribution by propidium iodide DNA staining through flow cytometry. In addition the expression of cyclins and cyclin-dependent kinases was evaluated by western blotting. A reduction of apigenin-induced cell growth was found in both cell lines, although SCC-25 cells were significantly more sensitive than the immortalized keratinocytes, HaCaT. Moreover, apigenin induced apoptosis and modulated the cell cycle in SCC-25 cells. Apigenin treatment resulted in cell cycle arrest at both G0/G1 and G2/M checkpoints, while western blot analysis revealed the decreased expression of cyclin D1 and E, and inactivation of CDK1 upon apigenin treatment. These results demonstrate the anticancer potential of apigenin in an oral squamous cell carcinoma cell line, suggesting that it may be a very promising chemopreventive agent due to its cancer cell cytotoxic activity and its ability to act as a cell cycle modulating agent at multiple levels.

Myricetin and Naringenin Inhibit Human Squamous Cell Carcinoma Proliferation and Migration In Vitro

In this study the potential anticancer effect of 2 flavonoids, myiricetin (MYR) and naringenin (NAR) has been evaluated on an oral squamous cell carcinoma (OSCC) cell line, SCC-25, and HaCaT cells. Both the flavonoids inhibited SCC-25 cell growth, although NAR selectively affected cancer cells without impairing HaCaT cell growth. The cell proliferation inhibition by MYR and NAR was not related to apoptosis induction, but on cell cycle impairment, because a G0/G1 and a G2/M blockage was highlighted following 24 h of treatment in SCC-25 and HaCaT cells, respectively. Western blot analysis showed that MYR induced a decrease of Cyclin D1 in SCC-25 and of Cyclin B1 in HaCaT cells, while NAR negatively modulated Cyclin D1 expression in SCC-25 cells. Wound-healing and cell invasion assays demonstrated that both the flavonoids were able to reduce motility on both SCC-25 and HaCaT cells. In conclusion the results of the present study show the anticancer potential of NAR and MYR on OSCC because they exert cytostatic effect by the impairment of cell cycle progression. Moreover both the flavonoids inhibit cell migration, thus highlighting their potential effect as antimetastatic agents. Therefore, MYR and NAR appear as promising candidate as oral cancer chemopreventive agents.

Flavonoids in oral cancer prevention and therapy.

Oral cancer, representing all the malignancies arising in the oral cavity, is the eighth most diffused neoplasm worldwide. Despite therapeutic improvements, its survival rate has not changed significantly over the past few decades, with a 5-year survival rate slightly above 50%. In this context, a search for new therapeutic strategies is mandatory. Flavonoids, polyphenolic compounds derived from plants, have a broad spectrum of biological activities, including antioxidant and anticancer. They have been proved to counteract the growth of several types of cancer through multiple mechanisms including the inhibition of cell cycle progression, apoptosis induction, and the modulation of intracellular pathways. Because of their multiple biological activities and their safe toxicological profile, flavonoids have been studied widely in the last decade as potential leads for anticancer therapy. Several studies have reported different flavonoid effects according to cancer cell type. In the present review, therefore, we have evaluated the data available on the effect of flavonoids on oral cancer, with the aim of identifying the molecular mechanisms underlying their potential anticancer properties.

Association between metalloproteinases 2 and 9 activity and ERK1/2 phosphorylation status in head and neck cancers: An ex vivo study

Advances in clinical treatment of head and neck squamous cell carcinoma (HNSCC) are hampered by its high infiltrative potential leading to distal metastasis. Since their ability to degrade the basal lamina and extracellular matrix, matrix metalloproteinases (MMP) have a pivotal role in tumor invasion. The overexpression and the aberrant activity of MMPs especially of MMP2 and MMP9, during HNSCC development and progression have been reported. However, up to now little is known about the mechanism of their regulation in HNSCC. It has been demonstrated that MMP2/9 expression is negative regulated by extracellular signal regulated kinase 1 and 2 (ERK1/2) in HNSCC cell lines. ERKs are protein kinases belonging to the mitogen-activated protein kinases family, and they are involved in the regulation of different cellular aspects, from apoptosis to cell proliferation and differentiation. In the present study we evaluated MMP2 and MMP9 activity by gelatine zymography in 16 tissue samples of HNSCC and their paired normal mucosa from patients undergoing surgical treatment. Moreover, ERK1/2 activation was analyzed by immunoblotting. A statistically significant decrease in the levels of activated ERK2 in cancer specimens in comparison with paired normal tissues was observed, whereas a significant increase in the activity of MMP2 was found in cancer specimens. However, the statistical analysis failed to demonstrate a correlation between the increase in MMP2 activity and the reduction of ERK1/2 activation levels. The results obtained, therefore, rule out, for the first time in an ex vivo study, the existence of a negative correlation between ERK1/2 activation and MMP2 activity.

Different Effects of Erythropoietin in Cisplatin- and Docetaxel-Induced Neurotoxicity: An In Vitro Study

Chemotherapy‐induced peripheral neurotoxicity (CIPN) is a side effect limiting cisplatin (CDDP) and docetaxel (DOCE) treatment. Erythropoietin (EPO) is a hematopoietic growth factor also displaying neurotrophic properties. Evidence suggests that EPOs neuroprotective action may rely on PI3K/AKT pathway activation; however, data regarding the EPO neuroprotective mechanism are still limited. This study evaluated the effect of EPO on organotypic cultures of rat dorsal root ganglia (DRG) and in primary cultures of DRG‐dissociated sensory neurons exposed to CDDP‐ and DOCE‐induced neurotoxicity, aiming to investigate EPOs neuroprotective mechanism. Subsequently, the levels of AKT expression and activation were analyzed by Western blot in neurons exposed to CDDP or DOCE; AKTs role was further evaluated by using a chemical inhibitor of AKT activation, wortmannin. In these models EPO, was protective against both CDDP‐ and DOCE‐induced cell death and against CDDP‐induced neurite elongation reduction. A modulation of AKT activation was observed in CDDP‐treated neurons, and the presence of wortmannin prevented EPOs neuroprotective action against CDDP toxicity but did not have any effect on EPOs protection against DOCE‐induced toxicity, thus ruling out the PI3K‐AKT pathway as the mechanism of EPOs effect in neuronal death prevention after DOCE exposure. Our results confirm in vitro the effectiveness of EPO as a neuroprotectant against both CDDP‐ and DOCE‐induced neurotoxicity. In addition, a role of PI3K/AKT in EPOs protection against CDDP, but not against DOCE, neurotoxicity was shown, suggesting that alternative pathways could be involved in EPOs neuroprotective activity.

Expression, distribution and glutamate uptake activity of high affinity-excitatory aminoacid transporters in in vitro cultures of embryonic rat dorsal root ganglia

Glutamate is the major mediator of excitatory signalling in the mammalian central nervous system, but it has recently been shown to play a role in the transduction of sensory input at the periphery and in peripheral neuropathies. New advances in research have demonstrated that rat peripheral sensory terminals and dorsal root ganglia (DRG) express molecules involved in glutamate signalling, including high-affinity membrane-bound glutamate transporters (GLAST [glutamate aspartate transporter], GLT1 [glutamate transporter 1], EAAC1 [excitatory aminoacid transporter 1]) and that alterations in their expression and/or functionality can be implicated in several models of peripheral neuropathy, neuropathic pain and hyperalgesia. Here we describe, through immunoblotting, immunofluorescence assays and β-counter analysis of [(3)H] l-glutamate uptake, the expression, distribution and activity of the glutamate transporters in in vitro cultures of embryonic dorsal root ganglia sensory neurons, sensory neurons+satellite cells and satellite cells. In this work we demonstrated that glutamate transporters are expressed in all cultures with a peculiar pattern of distribution. Even if GLAST is strongly detected in satellite cells, it is slightly expressed also in sensory neurons. GLT1 immunostaining is very weak in DRG neurons, but it was evident in the satellite cells. Finally, EAAC1 is localized in the soma and in the neuritis of sensory neurons, while it is not detectable in satellite cells. Moreover, all the cell cultures showed a strong sodium-energy-dependent glutamate uptake activity and it is more marked in neurons alone or in co-culture with satellite cells compared to satellite cells alone. Finally, we show that the complete or partial pharmacological inhibition of glutamate transporters virtually completely or partially abolish glutamate uptake in all cell culture. These results, that demonstrate that functionally active glutamate transporters can be studied in dorsal root ganglia cell cultures, provide further evidence for a role of glutamatergic transport in the peripheral nervous system and will be useful for testing whether any changes occur in in vitro models of peripheral nervous system damage.

Neurobasal medium toxicity on mature cortical neurons.

Neurobasal medium (NBM) is a widely used medium for neuronal cultures, originally formulated to support survival of rat hippocampal neurons, but then optimized for several other neuronal subtypes. In the present study, the toxic effect of NBM on long-term cortical neuron cultures has been reported and investigated. A significant neuronal cell loss was observed 24 h after the total medium change performed at days in vitro 10. The neurotoxic effect was specifically because of NBM-A, a commercially derived modification of classic NBM, as neurons exposed to minimum essential medium for 24 h did not show the same mortality rate. We showed that the toxic effect was mediated by the N-methyl-D-aspartate receptor (NMDAr) as its inactivation partly prevented NBM-induced neuronal loss, and the addition of NMDAr activators, such as L-cysteine or glycine to minimum essential medium, reproduced the same toxicity rate observed in NBM. Besides the toxicity associated with NMDAr activation, the decreased antioxidative defenses also worsen (because of glutathione depletion) neuronal death, thus amplifying the effect of excitotoxic amino acids. Indeed, glutathione supplementation by the addition of its precursor N-acetyl-cysteine resulted in an increase in neuronal survival that partially prevented NBM-A toxicity. These results evidenced, on the one hand, the unsuitability of NBM-A for long-term neuronal culture, and on the other, they highlight the importance of selection of more suitable culture conditions.

Undifferentiated MSCs are able to myelinate DRG neuron processes through p75

Over the last few years the therapeutic approach to demyelinating diseases has radically changed, strategies having been developed aimed at partnering the classic symptomatic treatments with the most advanced regenerative medicine tools. At first, the transplantation of myelinogenic cells, Schwann cells or oligodendrocytes was suggested, but the considerable technical difficulties, (poor availability, difficulties in harvesting and culturing, and the problem of rejection in the event of non-autologous sources), shifted attention towards more versatile cellular types, such as Mesenchymal Stem Cells (MSCs). Recent studies have already demonstrate both in vitro and in vivo that glially-primed MSCs (through exposure to chemical cocktails) have myelogenic abilities. In spite of a large number of papers on glially-differentiated MSCs, little is known about the ability of undifferentiated MSCs to myelinate axons and processes. Here we have demonstrated that also undifferentiated MSCs have the ability to myelinate, since they induce the myelination of rat DRG neuron processes after direct co-culturing. In this process a pivotal role is performed by the p75 receptor.

Comparing the different response of PNS and CNS injured neurons to mesenchymal stem cell treatment

Abstract Mesenchymal stem cells (MSCs) are adult bone marrow‐derived stem cells actually proposed indifferently for the therapy of neurological diseases of both the Central (CNS) and the Peripheral Nervous System (PNS), as a panacea able to treat so many different diseases by their immunomodulatory ability and supportive action on neuronal survival. However, the identification of the exact mechanism of MSC action in the different diseases, although mandatory to define their real and concrete utility, is still lacking. Moreover, CNS and PNS neurons present many different biological properties, and it is still unclear if they respond in the same manner not only to MSC treatment, but also to injuries. For these reasons, in this study we compared the susceptibility of cortical and sensory neurons both to toxic drug exposure and to MSC action, in order to verify if these two neuronal populations can respond differently. Our results demonstrated that Cisplatin (CDDP), Glutamate, and Paclitaxel‐treated sensory neurons were protected by the co‐culture with MSCs, in different manners: through direct contact able to block apoptosis for CDDP‐ and Glutamate‐treated neurons, and by the release of trophic factors for Paclitaxel‐treated ones. A possible key soluble factor for MSC protection was Glutathione, spontaneously released by these cells. On the contrary, cortical neurons resulted more sensitive than sensory ones to the toxic action of the drugs, and overall MSCs failed to protect them. All these data identified for the first time a different susceptibility of cortical and sensory neurons, and demonstrated a protective action of MSCs only against drugs in peripheral neurotoxicity. HighlightsCNS neurons and PNS neurons have a different susceptibility to neurotoxic drugs.Direct contact and soluble factor release were involved in MSCs’ protective effect.MSCs protect sensory neurons but fail to protect cortical neurons from drugs toxicity.

T-helper and T-regulatory cells modulation in head and neck squamous cell carcinoma

ABSTRACT Head and neck squamous cell carcinoma (HNSCC) is one of the most diffused cancer types, characterized by a high reoccurrence rate, mainly due to the inability of current therapeutic approaches to completely eradicate cancer cells. HNSCC patients often have defective immune functions, thus allowing cancer immune escape and cancer spreading. Particularly important in driving immune escape during HNSCC progression are T-helper and T-regulatory cells. New insights into their mechanisms of action might support the development of effective and long-lasting immunotherapy.