Nitric oxide action on growth factor-elicited signals. Phosphoinositide hydrolysis and [Ca2+]i responses are negatively modulated via a cGMP-dependent protein kinase I pathway.

Abstract The role of nitric oxide (NO) in the phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and intracellular Ca2+ release responses induced by epidermal, platelet-derived, and fibroblast growth factors was investigated in three cell lines, a clone of NIH-3T3 fibroblasts overexpressing epidermal growth factor receptors and the tumoral epithelial cells A431 and KB. In all three cell types, pretreatment with NO donors decreased growth factor-induced PIP2 and Ca2+ responses, whereas pretreatment with NO synthase inhibitors increased them. The Ca2+-dependent PIP2 hydroysis induced by micromolar concentrations of the Ca2+ ionophore, ionomycin, was also modulated negatively and positively by NO donors and synthase inhibitors, respectively. In contrast, the Ca2+ content of the intracellular stores was unaffected by the various pretreatments employed. NO donors and synthase inhibitors induced an increase and decrease, respectively, of the intracellular cGMP formation in all three cell lines investigated. All of the effects of the NO donors were mimicked by 8-bromo-cGMP administration and abolished by pretreatment with the specific blocker of the cGMP-dependent protein kinase I, KT5823, which by itself mimicked the effects of the synthase inhibitors. Together with previous observations on G protein-coupled receptors, the present results demonstrate that PIP2 hydrolysis and Ca2+ release occur under the feedback control of NO, independently of the phospholipase C (β, γ, or δ type) involved and of the mechanism of activation. Such a control, which appears to be effected by the cGMP-dependent protein kinase I acting at the level of the phospholipases C themselves, might ultimately contribute to the inhibitory role of NO on growth previously observed with various cell types

Embryonic rat dorsal root ganglia organotypic culture: a morphometric model to test neurotoxicology

Neurotoxicity is a common dose-limiting side-effect of several drugs (Cavaletti et al., 2008). So far a validated test method to screen drugs neurotoxicity does not exist, therefore in this interdepartment study we have analyzed the effectiveness of a morphometric neurotoxicty assessment model. Drug neurotoxicity evaluation is based on embryonic rat dorsal root ganglia (DRG) organotypic culture. DRG primary sensory neurons are the principal target of drugs neurotoxic action. In fact, primary sensory neurons lie outside the blood-nerve barrier and are supplied by capillaries with fenestrated walls. Moreover, the axons of these cells are among the longest of the entire nervous system and, therefore, are more susceptible to any agent that interferes with the energy metabolism or the structural basis of axonal transport. In particular, in this interdepartment study, the interference of the under study neurotoxic compound with NGF-induced neurite elongation is analysed. The effectiveness and reproducibility of this model, even if commonly used to test drugs, has not yet been demonstrated. In order to assess the validity of this in vitro model, antineoplastic drugs known to be in clinical use and in animal models neurotoxic (paclitaxel and oxaliplatin) or not dangerous (cyclophosphamide and 5-Fluorouracil) have been tested. DRGs explanted from E15 rat embryos have been treated for 24h with drugs concentrations comparable to those achievable in vivo. The length of the longest neurite of each DRG has been measured by ImageJ program. Experiments have been performed by three different blinded researchers in two different laboratories. Mean and standard deviation of each experiment were obtained, subsequently the mean value and standard deviation of the three independent experiments for each researcher were calculated. Data obtained by the three researchers in two different laboratories resulted statistically comparable and no significant differences were detected (One Way Anova analysis of variance and Tukey post test; p<0.05). This interdepartment in vitro study, therefore, indicates that a purely morphometric model represents a reliable tool to study drug neurotoxicity, permitting to make prediction of neurotoxic effects on humans because the concentrations tested are the same to which DRG are exposed during clinical use

The fundamental role of morphology in experimental neurotoxicology: the example of chemotherapy-induced peripheral neurotoxicity

The peripheral nervous system is a frequent target of toxic agents. The accurate identification of the sites of neurotoxic action through the morphological characterization of reliable in vivo models or in vitro systems can give fundamental clues when investigating the pathogenesis and interpreting the clinical features of drug-induced neuropathy. The morphological approach has been used to investigate almost all the anticancer drugs able to induce chemotherapy-induced peripheral neurotoxicity, i.e. platinum drugs, antitubulins and proteasome inhibitors. No models have ever been described for thalidomide. This review demonstrates that any pathogenetic study on chemotherapy-induced peripheral neurotoxicity must be based on solid morphological observations obtained in reliable animal and in vitro models. This is particularly true in this setting, since the availability of tissues of human origin is extremely limited. In fact, peripheral (generally sural) nerve biopsies are never required for diagnostic purposes in chemotherapy-treated cancer patients, and their use for a purely scientific aim, although potentially very informative, is not ethical. Moreover, several neurotoxic drugs target the dorsal root ganglia neurons, and it is very difficult to obtain high-quality specimens even from early autopsies. It is, therefore, our opinion that an extensive morphological assessment of the in vitro and in vivo effect of any potentially neurotoxic antineoplastic drugs, as well as of neuroprotectant agents, should be taken into consideration right from the earliest stages of their development

Antitumoral effects of Hibiscus Sabdariffa on human breast cancer cells

Hibiscus Sabdariffa (HS) is a plant commonly used in folk medicine (1). In recent years HS has gained great interest due to its important antioxidant, anti-inflammatory and antitumoral properties. In our work, we evaluated the in vitro anticancer effects of HS extract against two different human breast cancer cell lines: estrogen receptor (ER) positive MCF-7 cells and ER negative MDA-MB-231 cells. We tested both total extract (HSE) and one fraction obtained by ethyl acetate extraction (HSEC). MTT assay and Trypan Blue vital count showed a dose and time dependent reduction of the viability in both cell lines treated with different concentrations of HSE or HSEC compared to untreated control cells. A significantly marked reduction was observed in MCF-7 cells treated with HSEC. On the basis of our results we used the concentrations of 7.5mg/ml and 3.5mg/ml respectively for HSE and HSEC. In order to evaluate ER involvement in HS effect, we analyzed the cellular localization of the receptor (ERα isotype) by immunofluorescence experiments. Untreated MDA-MB-231 cells showed a low expression of the receptor mostly localized at the cytoplasmic level and treatment with HSE or HSEC didn’t change this state. Untreated MCF-7 cells showed a greater expression of the receptor, with nuclear and cytoplasmic localization. Following HSE or HSEC treatment ERα localization became more cytoplasmic and this effect was more evident after HSEC induction. These data were also confirmed by ERα western blot analysis. Subsequently, we studied HSE and HSEC ability to alter migration and invasion capacity of ER positive MCF-7 cells. Using a scratch wound healing assay we did not observe any change in the migration of cells compared to untreated cells. On the contrary, in a Boyden chamber invasion assay, HSE, and especially HSEC, induced reduction of MCF-7 cell invasion. In conclusion, we have demonstrated that HS is able to reduce cell viability of ER positive MCF-7 and ER negative MDA-MB-231 cells. This effect is more evident in MCF-7 cells in which ER localization and reduced cell invasion were observed. These results are more evident after HSEC treatment. Further studies will be needed to better elucidate the involved mechanisms of action

Human oral squamous cell carcinoma proliferation and migration prevented by two flavonoids

Oral Cancer (OC) is one of the most frequent cancer in Head and Neck district and Oral Squamous Cell Carcinoma (OSCC) constitutes the large majority of the neoplasia arising in oral cavity. OSCC remains a hampering matters for clinics, since the overall disease free survival has not significantly increased during the last decades and invasion to surrounding tissue and to regional lymph nodes is often reported. Therefore new strategies to prevent and inhibit OSCC growth and invasion are highly desirable and new therapeutic approaches are currently tempted also with the use of natural compounds. Myricetin (MYR) and Naringenin (NAR), two naturally occurring flavonoids, widely diffused in plants, fruits and vegetable, have recently gained consideration thanks to their anti oxidant, anti inflammatory and anti tumoral properties. In this study their potential anticancer effect has been evaluated on an OSCC cell line, SCC-25 and on spontaneously immortalized non tumoral keratinocytes, HaCaT cells. MYR and NAR induce a significant cell growth inhibition in SCC-25 cells, in addition NAR selectively affected cancer cells, since it does not impair HaCaT cell growth. Furthermore an additive effect of MYR and NAR has been highlighted. The cell proliferation inhibition is not related to apoptosis induction, as demonstrated by evaluation of phosphatidyl serine membrane translocation and dapi staining. On the contrary MYR and NAR effect depends on the cell cycle progression impairment. Wound-healing and cell invasion assays, respectively performed by cell monolayer scratch and Boyden Chamber transwell test, demonstrate that the two flavonoids are able to reduce motility and invasiveness 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, since both flavonoids prevent cancer cell proliferation through a cytostatic effect, by the impairment of cell cycle progression. Moreover both the flavonoids inhibit cell migration, thus highlighting their potential effect as anti metastatic agents

Human Mesenchymal Stem Cells and Endothelial Progenitor Cells exert a neuroprotective effect on rat cortical neurons injured by oxygen and glucose deprivation

Oxygen and glucose deprivation (OGD) due to ischemic events or trauma in the brain result in neuronal loss. The therapeutic approaches available inadequate and often the outcome is unfavorable for the patient or at least unpredictable. Stem cells could be useful for the treatment of OGD injured-neurons. Mesenchymal Stem Cells (MSCs), isolated from bone marrow as well as from various tissues, have poor immunogenicity and neuroprotective properties being able to alleviate ischemic brain injuries in animal models. The Endothelial Progenitor Cells (EPCs) are present at low frequencies both in the bone marrow and in the peripheral blood. They are thought to play a role in the recovery of cerebrovasculature integrity after stroke. In the present study we evaluated the potential neuroprotective effect of human MSCs and human EPCs on rat embryonic cortical neurons injured by OGD. OGD was induced by incubating the cortical neurons in a hypoxia chamber in a 95% N2 + 5% CO2 atmosphere at 37°C without glucose. To set up the experimental protocol, OGD was maintained for 1, 2 and 3 hours. The neurons were returned in normoxic atmosphere and after 2 and 5 days neuronal survival was evaluated by MTT assay, LDH assay and viable cellular counting. The 2 hours OGD was able to reduce neuronal viability by 50% and was chosen for the subsequent experiments. To assess MSCs and EPCs neuroprotective action, after 2 hours-long OGD the neurons were 1) co-cultured with either MSCs or EPCs seeded on a cell culture insert avoiding direct contact while sharing the same medium, or 2) cultured in a medium previously conditioned by either MSCs or EPCs. Neuronal survival was evaluated by MTT assay after 2 and 5 days. Both MSCs and EPCs increased neuronal survival after ODG. The effect was observed in absence of a direct contact between MSCs or EPCs and the injured neurons, suggesting that the release of soluble factors may be involved in their neuroprotective action. In conclusion both MSCs and EPCs could represent a potential therapeutic approach for the treatment of brain ischemic injury. Further studies are needed to identify the specific molecules and pathways that play a role in the neuroprotective effect of MSCs and EPCs

Innovative 3D proteome-wide scale identification of ALKBH5 target for MV1035 small molecule able to reduce migration and invasiveness in U87 glioblastoma cell lines by SPILLO-PBSS

The innovative in silico technologies developed at SPILLOproject,1 e.g., the SPILLO potential binding sites searcher (SPILLO-PBSS) software,2,3 allow to identify targets and off-targets of any small molecule on a multiple-organism proteomewide scale, and to perform an accurate multilevel cross-organism transferability analysis (MCOTA) aimed at rationalising animal testing. SPILLO-PBSS has been successfully used in several research projects, such as a study in which a compound (MV1035) was found to reduce migration and invasiveness in U87 glioblastoma (GBM) cell lines: the human structural proteome was analyzed and the RNA demethylase ALKBH5 has been identified as a target responsible for the observed effects (target experimentally validated). Another top-ranked target identified by SPILLO-PBSS, the DNA repair protein AlkB homolog 2 (ALKBH2), abundantly expressed in GBM cell lines, resulted particularly interesting for its pivotal role in the onset of resistance to Temozolomide (TMZ), the standard firstline treatment for GBM.