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

Effects of valproic Acid, berberin and resveratrol on human mesenchymal stem cells adipogenic differentiation

Nowadays obesity and its related diseases represent a major health problem with an increasing worldwide prevalence. Hyperplasia and hypertrophy of adipocytes lead to an excessive fat accumulation that is not efficiently prevented by current pharmacological treatments. So the research on anti-obesity drugs with good efficacy and tolerability able both to prevent and to reduce fat accumulation is of pivotal interest. In the present study we evaluated in vitro the effects of Valproic Acid, Berberin and Resveratrol on adipogenesis. Our experimental model was represented by human Mesenchymal Stem Cells (hMSCs), physiological precursors of adipocytes that can differentiate into adipocytes also in vitro. Preliminary cytotoxicity assays were performed in order to choose non-toxic doses of the three drugs. hMSCs were induced to adipogenic differentiation and treated with Valproic Acid, Berberin and Resveratrol at the selected doses. Controls were represented by hMSCs treated for adipogenesis in absence of the drugs. At different time points intracellular lipid droplets accumulation, a typical feature of adipogenesis, was assessed by Oil Red O staining. Valproic Acid, Berberin and Resveratrol inhibited hMSCs adipogenic differentiation in a dose dependent manner as demonstrated by the reduction of the lipid droplets accumulation. To understand the molecular mechanisms of the drugs-induced adipogenesis inhibition, we focused our attention on the effects of the drugs treatment on cell cycle progression, known to be altered by many antiadipogenic drugs, and on the MAP Kinases ERK1 and ERK2, involved in the adipogenesis control. We evaluated the expression of cyclins and CDKs by immunoblotting and flow-cytometry analyses, demonstrating that Valproic Acid, Berberin and Resveratrol interfere on cell cycle progression. The expression and the phosphorylation status of the two kinases ERK1 and ERK2 were assessed by immunoblotting demonstrating an increase of ERK1 phosphorylation (i.e. activation) in hMSCs treated with Berberin and a reduction in hMSCs treated with Valproic Acid and Resveratrol compared to control cells. No changes in phosphorylation and expression of ERK2 were observed. Our study demonstrate that Valproic Acid, Berberin and Resveratrol exert an anti-adipogenic effect in our experimental model. The mechanisms of action of these drugs involve the alteration of cell cycle progression and, at least in part, ERK1/2 modulation. However other molecular pathways are likely implicated and other studies are required to identify them

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

Glutamate is the major mediator of excitatory signalling in the mammalian central nervous system, but it has recently been shown to play also 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 (Excitatory Aminoacid Transporters, EAATs) and that alterations in their expression and/or functionality can be implicated in several models of peripheral neuropathy, neuropathic pain and hyperalgesia. Since EAATS might represent an interesting target for pharmacological intervention, the knowledge of their distribution and functionality deserves to be improved. Here we describe, through immunofluorescence assays, immunoblotting and beta-counter analysis of (H3) L-glutamate uptake, the expression, distribution and activity of the EAATs in in vitro cultures of embryonic DRG sensory neurons, sensory neurons+satellite cells and satellite cells. In this study we demonstrated that EAATs are expressed in all cultures, but that their distribution recognizes a peculiar pattern for each of them, since EAATs immunolabelling was differentially expressed in the cytoplasm of neuronal or satellite cells. This result was further confirmed by immunoblotting. Moreover, both cell types showed a strong sodium-ATP-dependent (active) glutamate uptake activity. However, the net (i.e. active transport minus passive diffusion) glutamate transport was more marked in neuronal cultures when cells were grown and maintained without satellite cells. These results, that demonstrate that functionally active EAATs can be studied in DRG cell cultures, provide further evidence for a role of glutamatergic transport in the peripheral nervous system and will be useful for testing whether any change occurs in in vitro models of peripheral nervous system damage. This work was supported in part by an unrestricted research grant from the “Fondazione Banca del Monte di Lombardia”

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

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

miR-192-5p and CXCL2/NOD2 inverse correlation in celiac disease

Celiac disease (CD) is an inflammatory disorder of the small Intestine activated in genetically predisposed individuals by the ingestion of gluten from wheat and related cereals (1). The role of adaptative immunity in CD has been widely investigated. However, also innate immunity can have a role in the development of the disease, and it could involve the alteration of proteins expression (determined either at transcriptional and post-transcriptional level). Post-transcriptional regulation could involve also small non coding RNAs (micro RNA, miRNA). In this study we have analyzed miRNAs expression in duodenal biopsies of controls and celiac patients subdivided into 2 groups (Marsh 3A-B and Marsh 3C) according to the severity of the intestinal lesion. Microarray analysis demonstrated that miR-192-5p is significantly down-regulated in biopsies of Marsh 3C CD patients. Several studies suggested a possible role for miR-192-5p in diseases with alteration of the immune system (2, 3). In this study we identified possible target genes by in silico analysis, including CXCL2 and NOD2, molecules involved in innate immunity. qRT-PCR and western blotting demonstrated that both mRNA and protein expression were higher in CD patients compared to controls. qRT-PCR performed after laser microdissection and immunohistochemistry prove that miR-192-5p and CXCL2 and NOD2 mRNA were present principally in villus epithelium (both in controls and in CD patients) suggesting a direct interaction between miR-192-5p and the two target proteins. In conclusion the results of the present study support the role of miRNAs in regulating the innate immunity in celiac disease

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

Antitumoral effects of Hibiscus sabdarifa on human oral squamous cell carcinoma and multiple myeloma cells

Epidemiological data consistently demonstrate a reduced cancer risk associated with a polyphenols rich diet. Hibiscus sabdarifa (HS), a polyphenols rich plant widely consumed worldwide as beverage and used in folk medicine, has recently gained interest thanks to its antioxidant, anti-inflammatory and chemopreventive properties. In the present study we investigated the antitumoral potential of HS extract in two different human tumor cell lines: Multiple Myeloma cells (RPMI 8226) and Oral Squamous Cell Carcinoma cells (SCC-25). MTT assays showed that HS extract induced a dose-dependent viability reduction in both the cells lines. For the subsequent experiments we used HS at the concentration of 5 mg/ml that was the most effective in inducing cell viability reduction after 48h of treatment. Viable cell count using trypan blue staining demonstrated that the HS extract induced decrease in cell growth of both the cell lines and this was due to a reversible cytostatic rather than a cytotoxic effect. Wound-healing and cell invasion assays, respectively performed by a scratch of cell monolayer and Boyden Chamber transwell test, demonstrated that HS extract was able to reduce motility and invasiveness in both RPMI 8226 and SCC-25 cells. The chemical inhibition of ERK1/ERK2 and PI3K, with U0126 and wortmannin respectively, reduces proliferation and migration of both SSC-25 and RPMI cells and HB extract treatment played an additive action with the inhibitors. In conclusion, our results suggest that HS extract have antitumoral properties, since it proved to inhibit tumoral cell growth and cell migration and invasiveness. It is interesting to note that HS extract is effective against two very different tumor cell lines. In fact, RPMI 8226 cells are of hematopoietic origin and grow in suspension, whereas SCC-25 cells derive from epithelium and are characterized by adherent cell growth. Therefore, although further studies are needed to clarify the molecular mechanisms involved in its action, we proposed HS as a potential chemopreventive agent

Human Cutaneous Melanomas Lacking MITF and Melanocyte Differentiation Antigens Express a Functional Axl Receptor Kinase

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