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Additional file 1. Supplementary Methods.

Additional file 2: Supplementary Figs. S1 to S21. Supplementary Fig. S1. Expression in ten melanoma cell lines of genes and gene families targeted by epigenetic drugs. Supplementary Fig. S2. Differentiation profile of melanoma cell lines. Supplementary Fig. S3. Susceptibility of ten melanoma cell lines to the anti-proliferative effects of epigenetic drugs. Supplementary Fig. S4. Methylation status of LINE-1, MAGE-A1 and NY-ESO-1 in 9 melanoma cell lines treated with guadecitabine. Supplementary Fig. S5. Volcano plot of differentially expressed genes in VRG100 and CST30 cell lines. Supplementary FigureS6. Whole genome gene modulation analysis by 4 epigenetic drugs in two melanoma cell lines. Supplementary Fig. S7. Edwards-VENN diagram analysis of significantly modulated genes by epigenetic drugs. Supplementary Fig. S8. Original and revised gene classification of the NanoString nCounter PanCancer Immune Profiling panel. Supplementary Fig. S9. Quantitative analysis of Nanostring data in ten melanoma cell lines treated with epigenetic drugs. Supplementary Fig. S10. Modulation of immune-related genes in melanoma cell lines by epigenetic drugs. Supplementary Fig. S11. Comparison of immune-related gene modulation by BET inhibitors JQ1 and OTX-015. Supplementary Fig. S12. Immune-related signature of epigenetic drugs in melanoma. Supplementary Fig. S13. Outline of the strategy for quantitative analysis and visualization of western blot data. Supplementary Fig. S14. Quantitative western blot analysis and visualization of the modulation of LMP7 by epigenetic drugs in 11 melanoma cell lines. Supplementary Fig. S15. Pipeline of data analysis based on Upstream Regulators (UR) identified by IPA. Supplementary Fig. S16. Classification of URs significantly modulated by at least two different drugs in melanoma cell line VRG100. Supplementary Fig. S17. Classification of URs significantly modulated by at least two different drugs in melanoma cell line CST30. Supplementary Fig. S18. Summary of the major functional networks linking relevant URs emerging from IPA Core Analysis in melanoma cell line VRG100. Supplementary Fig. S19. Comparison of URs activated by guadecitabine in-vitro and in-vivo. Supplementary Fig. S20. Comparison of URs activated by guadecitabine in melanoma cell line VRG100 vs a mesothelioma cell line vs hepatocarcinoma cell lines. Supplementary Fig. S21. Prognostic significance of selected genes in the guadecitabine signatures in 41 TCGA tumor types.

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., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

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., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Cisplatin is one of the most effective metal-based anticancer agents, targeting a large number of solid tumours. Despite its efficacy, cisplatin treatment is still limited by severe side effects such as neuro-, hepato- and nephro-toxicity and by resistance phenomena, only partially overcome by the use of new platinum drugs (i.e. oxaliplatin and carboplatin). These problems have stimulated the research and development of alternative therapeutic strategies based on different heavy metals. In this work we investigated the in vitro activity and neurotoxicity of three anticancer complexes: [Cu(PTA)4]PF6, [Cu(thp)4]PF6 and [Au(PTA)4]PF6. Neurotoxicity was evaluated by embryonic rat dorsal root ganglia (DRG) organotypic culture model. Furthermore the extent of proteasome inhibition in rat embryonic DRG neurons was evaluated by fluorimetric assay. After 48 hours of treatment, both copper-based compounds were not neurotoxic even at higher concentrations with respect to the IC50 obtained in A549 and IGROV-1 human cancer cells while [Au(PTA)4]PF6 was neurotoxic at lower concentration than IC50 in cancer cell lines tested. Since the ubiquitin-proteasome system has been identified as molecular target in cancer cells for the heavy metal based-drug, we evaluated their ability to affect the proteasome machinery in DRG neurons. The copper-based compounds, that are not neurotoxic, do not inhibit proteasome activity in DRG neurons. On the contrary, the neurotoxic complex [Au(PTA)4] PF6, induces a significant inhibition of proteasome activity even at concentration lower than the IC50. Furthermore, based on the content in heavy metal-based atoms, the complexes used in this study are suitable candidates for Photon Activation Therapy (PAT). Pretreatment of IGROV-1 cells with [Cu(PTA)4]PF6 induces an increase cell death with respect to drug or synchrotron (SR) alone. Furthermore the SR/[Cu(PTA)4]PF6 combinational treatment induced an increase in DNA damage with respect to single treatments. Our results, together with the low IC50 of the copper compounds compared to the one observed for cisplatin, suggest them as promising compounds in anticancer treatment., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

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., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

Background. Glioblastoma multiforme (GBM) is one of the deadliest cancers since it shows a very limited sensitivity to chemo- and/or radiotherapy. Experimental evidence suggests that the presence in the tumour environment of GBM stem-like cells might be particularly relevant to confer this extraordinary resistance to treatment. Aim. In this study, we aimed at providing a proof-of-concept of the efficacy of Photo Activation Therapy (PAT) using monochromatic X-rays, issued from a synchrotron radiation (SR) source, in killing human glioma stem cells pre-treated with cisplatin. We have also investigated if lethality of this treatment was superior to that achievable with conventional X-ray irradiation. Material and Methods. Cisplatin was chosen as the carrier of platinum atoms in the cells based on previous experience in glioma and non-glioma cancer cells; the irradiation was performed by delivering 1-8 Grays using beams of energies just above or below the platinum K-shell edge (78.39 keV) or with a conventional X-ray source. Cells were exposed to low drug concentrations allowing 90% or more of GBM stem-like cells survival thus mimicking the unfavourable tissue distribution generally achieved in GMB patients. Results. Under these experimental conditions a significant enhancement in cell lethality was observed using SR with respect to conventional X-ray irradiation. Conclusions. Our results provide additional support to the suggestion that PAT deserves to be further explored in relevant in vivo models based on GBM stem-like cells.

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., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Cisplatin (CDDP) is an anticancer drug widely used in clinic for the treatment of several solid tumours, despite its high effectiveness treatment is still limited by severe side effects and by inherited or acquired resistance phenomena. These drawbacks have stimulated the search of alternative strategies based on different metals offering a better toxicity profile while maintaining the same level of efficacy as platinum-based treatments. [1, 2] Recently we evaluated the cytotoxicity and neurotoxicity of our newly developed promising water soluble anticancer complexes ([Cu(PTA)4]PF6, [Cu(thp)4]PF6, [Au(PTA)4]PF6, [Au(thp)4]PF6) using CDDP as reference drug. The cytotoxicity was evaluated.on A549 non-small cell lung cancer (NSCLC) and IGROV-1 ovarian human cancer cells while the neurotoxicity was tested on dorsal root ganglia organotypic cultures. In our model CDDP resulted neurotoxic at concentrations achievable in plasma of patients treated with the same drugs. Similarly the gold-based compound [Au(PTA)4]PF6 was neurotoxic at lower concentration than IC50 calculated for the tested cancer cell lines. On the contrary, both copper-based compounds and [Au(thp)4]PF6 were neurotoxic at higher concentrations with respect to the IC50 obtained in tumour cell lines tested. We then tested at the ID17 beamline of the ESRF the efficacy of synchrotron radiation (SR) to trigger the Auger effect in IGROV-1 cells containing a high Z-number element. Irradiation of cells pre-treated with CDDP or [Cu(PTA)4]PF6 concentrations allowing roughly 90% of cell survival induced an enhancement in cellular death with respect to drug and irradiation alone. With the other compounds no cell death enhancement was observed. Our results suggest that SR-enhanced CDDP activity might allow the use of a reduced dose of CDDP thus achieving side effects minimization due to the exposure of normal cells/tissues to less toxic doses. Furthermore, considering the anticancer activity and the neurotoxic profile of [Cu(PTA)4]PF6, our data suggest that copper-based drugs represent new and promising compounds in anticancer treatment also in combination with SR. Acknowledgments Authors thanks the COST action TD1205 References: [1] Ceresa, C.; Bravin, A.; Cavaletti, G.; Pellei, M. and Santini C. Curr. Med. Chem. 2014, 21, 2237-2265. doi: 10.2174/0929867321666140216125721 [2] Ceresa, C; Nicolini, G; Semperboni, S; requardt, H; Le Duc, G; Santini, C; Pellei, M; Bentivegna, A; Dalprà, L; Cavaletti, G; Bravin, A. Anticancer Res. 2014, in press.

Background. Cisplatin is one of the most efficient metal-based anticancer agents, targeting several solid tumours. Despite its efficacy, cisplatin treatment is still limited by severe side effects such as neuro-, hepato- and nephro-toxicity and by resistance phenomena, only partially overcome by the use of new platinumdrugs (i.e. oxaliplatin and carboplatin). These problems have stimulated the research and development of alternative therapeutic strategies based on different heavy metals. Furthermore, heavy metal-based anticancer complexes represent suitable candidates to be used in Photon Activation Therapy (PAT). Aim. We investigated the in vitro activity and neurotoxicity of three anticancer complexes: [Cu(PTA)4]PF6, [Cu(thp)4]PF6 and [Au(PTA)4]PF6. Furthermore we investigated the efficacy of synchrotron radiation (SR) to trigger the Auger effect in human IGROV-1 ovarian cancer cells pre-treated with [Cu(PTA)4]PF6. Material and Methods. Neurotoxicity was evaluated by embryonic rat dorsal root ganglia (DRG) organotypic culture model. Furthermore the extent of proteasome inhibition in rat embryonic DRG neurons was evaluated by fluorimetric assay. The efficacy of SR in combination with [Cu(PTA)4]PF6 was evaluated, in terms of cell survival, by standard clonogenic assay. DNA damage induced by the combinational treatment was also tested by RAD51 nucelar foci counting and BRCA1 nuclear expression. Results. After 48 hours of treatment, both copper-based compounds were not neurotoxic even at higher concentrations with respect to the IC50 obtained in A549 and IGROV-1 human cancer cells while [Au(PTA)4]PF6 was neurotoxic at lower concentration than IC50 in cancer cell lines tested. Since the ubiquitin-proteasome system has been identified as molecular target in cancer cells for the heavy metal based-drug, we evaluated their ability to hinder the proteasome machinery in DRG neurons. Both copper-based compounds, that are not neurotoxic, do not inhibit proteasome activity in DRG neurons. On the contrary, the neurotoxic complex [Au(PTA)4]PF6, induces a significant inhibition of proteasome activity even at concentration lower than the IC50. PAT experiment in IGROV-1 cells showed that the pre-treatment with [Cu(PTA)4]PF6 induces an increase in cell death with respect to drug or SR alone. Furthermore the SR/[Cu(PTA)4]PF6 combinational treatment induced an increase in DNA damage with respect to single treatments. Conclusions. Our results, together with the low IC50 of the copper compounds compared to the one observed for cisplatin, suggest them as promising compounds in anticancer treatment. Finally our results suggest the potential use of [Cu(PTA)4]PF6 in PAT providing support to further studies in reliable in vivo animal models.

Cisplatin (CDDP) is an anticancer drug widely used in clinic for the treatment of several solid tumours. Despite its high effectiveness CDDP treatment is still limited by severe side effects and by inherited or acquired resistance phenomena, only partially overcome by the use of new platinum drugs (i.e. oxaliplatin and carboplatin). These drawbacks have stimulated the search of alternative strategies based on different metals offering a better toxicity profile while maintaining the same level of efficacy as platinum-based treatments. In this study we evaluated the cytotoxicity and neurotoxicity of new promising water soluble anticancer complexes based on copper [Cu(PTA)4PF6] [Cu(thp)4][PF6] and gold [Au(PTA)4PF6] [Au(thp)4][PF6] on A549 non-small cell lung cancer (NSCLC) and IGROV-1 ovarian human cancer cells. IC50 at 48 hours of treatment was calculated by SRB assay while the neurotoxicity was tested evaluating the interference of the compounds with neurite elongation. CDDP was used as drug reference with respect to our newly developed drugs. In our model CDDP resulted neurotoxic at concentrations achievable in plasma of patients treated with the same drugs. Similarly the gold-based compound [Au(PTA)4PF6] was neurotoxic at lower concentration than IC50 calculated for the tested cancer cell lines. On the contrary, both copper-based compounds and [Au(thp)4][PF6] were neurotoxic at higher concentrations with respect to the IC50 obtained in tumour cell lines tested. We then tested the efficacy of synchrotron radiation (SR) to trigger the Auger effect in A549 and IGROV-1 cells containing a high Z-number element, like platinum, gold, and copper. CDDP and the new metal-based drugs were used as carrier of heavy Z-elements. The irradiation was performed with monochromatic beams above the K-shell edge of the different elements. Using keV X-rays, the high photoelectric cross sections of the heavy-Z elements result in substantial interactions, producing fluorescence light and free electrons. The experiments were carried out at the ID17 beamline of the European Synchrotron Radiation Facility, where a high-fluence monochromatic beam adjustable from 20 to 180 keV is available. Irradiation of cells pre-treated with CDDP or [Cu(PTA)4PF6] concentrations allowing roughly 90% of cell survival induced an enhancement in cellular death with respect to drug and irradiation alone. With the other compounds no cell death enhancement was observed. Finally we studied the homologous recombination repair pathways. SR in combination with CDDP resulted in a significative nuclear relocalization of RAD51 and BRCA1 proteins with respect to untreated control A549 and IGROV-1 cells as well as in cells treated with SR or CDDP alone. Similar results were observed in IGROV-1 cells pre-treated with [Cu(PTA)4PF6]. Our results suggest that SR-enhanced CDDP activity might allow the use of a reduced dose of CDDP thus achieving side effects minimization due to the exposure of normal cells/tissues to less toxic doses. Furthermore, considering the anticancer activity and the neurotoxic profile of [Cu(PTA)4PF6], our data suggest that copper-based drugs represent new and promising compounds in anticancer treatment also in combination with SR.

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, Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

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., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

Cisplatin is the archetypal inorganic drug and it is nowadays one of the most efficient metal-based anticancer agents, targeting a large number of solid tumours. Although highly effective in treating a variety of cancers, cisplatin treatment is still limited by severe side effects such as neuro-, hepato- and nephro-toxicity and by inherited or acquired resistance phenomena, only partially overcome by the use of new platinum drugs (i.e. oxaliplatin and carboplatin). These problems have stimulated the research and development of alternative therapeutic strategies based on different heavy metals. In this work we investigated the neurotoxicity effect of two new promising water soluble anticancer complexes based on copper [Cu(PTA)4PF6] and gold [Au(PTA)4PF6]. Cisplatin and oxaliplatin neurotoxicity was also measured as reference drugs. Neurotoxicity was evaluated by an in vitro model based on the ability of NGF exposed embryonic rat dorsal root ganglia (DRG) organotypic culture to grow neurite. In particular the interference of the under study neurotoxic compound with neurite elongation was analysed. DRGs explanted from E15 rat embryos were treated for 48h with different drug concentrations including the IC50 evaluated for different tumour cell lines. In our model cisplatin and oxaliplatin resulted neurotoxic (inducing a reduction of neurite outgrowth more than 50%) at concentrations achievable in plasma of patients treated with the same drugs confirming data obtained in clinical studies. On the contrary, the neurotoxicity assay demonstrated that copper-based compound was neurotoxic at higher concentrations with respect to the IC50 obtained for all tumour cell lines tested. On the other hand preliminary neurotoxicity data suggest that gold-based compound is neurotoxic at lower concentration than IC50 calculated for almost tested cancer cell lines. Our results, together with the low IC50 of the copper compound compared to the one observed for cisplatin and oxaliplatin, suggest that copper-based drugs represent new and promising compounds in anticancer treatment.

Cisplatin is an antineoplastic drug widely used in clinic for the treatment of several solid tumours. However, at times, the side effects related to cisplatin-based anticancer therapy often outweigh the benefits. Several of these side effects are now manageable with effective treatment planning and supportive care strategies (e.g. anti-emetics, hematopoietics growth factors, physical treatments, hyperhydration), but others are still unsolved, can be dose-limiting and represent an unmet clinical need, for instance nervous system damage. Therefore, the identification of new anticancer strategies able to offer a better toxicity profile maintaining the same level of efficacy as platinum-based treatments would be highly desirable. We tested the efficacy of synchrotron radiation to trigger the Auger effect in the living human A549 non-small cell lung cancer (NSCLC) and IGROV-1 ovarian cancer cells pre-treated with cisplatin. Furthermore we tested the anticancer treatment in human VIPI glioblastoma stem cells that seem to be one of the most important determinants in the extraordinary resistance of GBL to any treatment tested so far. The experiments were carried out at the ID17 beamline of the European Synchrotron Radiation Facility, where a high-fluence monochromatic beam adjustable from 20 to 80 keV is available. Cisplatin was chosen as the carrier of platinum atoms in the cells because of its alkylating-like activity and the irradiation was done with monochromatic beams above and below the platinum K-shell edge (78.39 keV). Control cell survival curves were comparable with those obtained for the same cells under conventional irradiation conditions. On cisplatin-treated cells at a concentrations inducing 80% of cell survival with respect to the control, no differences were observed in cell survival when cells were irradiated either above or below the K-shell edge of platinum, suggesting that cisplatin toxicity can mask the enhancement of cell death induced by the irradiation above the K-shell edge. At lower cisplatin concentrations, determining 95-90% of cell survival an enhancement in the cellular death was observed, with respect to conventional irradiation conditions was observed when cells were irradiated with beams either above or below the platinum K-shell edge. Our results suggest that SR-enhanced CDDP activity might allow the use of a reduced dose of CDDP thus achieving side effects minimization due to the exposure of normal cells/tissues to less toxic doses. Furthermore SR-enhanced CDDP activity might enhance CDDP effect on cancer stem cells obtained from human glioblastoma. Reliable in vivo animal models might be investigated in future experiments to assess activity and toxicity of the proposed treatment in cancer-bearing mice.

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”., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

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., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Neurotoxicity is a common dose-limiting side-effect of several drugs. 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 neurotoxicty assessment model. Drug neurotoxicity evaluation in this model is based on the ability of NGF exposed embryonic rat dorsal root ganglia (DRG) organotypic culture to grow neurite; in particular the interference of the under study neurotoxic compound with 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

Organic cation transporters (OCTs) play an important role in transporting cationic xeno- and endobiotics across biological membranes. In particular their role in platinum drugs transport, such as cisplatin and oxaliplatin, has recently been studied in many normal tissues and cancer cells. It has become increasingly clear the role of these transporters in key tissues responsible for drug absorption and disposition: kidney, liver and intestine. However, only limited information is available regarding their distribution and activity in nervous system. This study was aimed to investigate (1) the expression of OCT2 in rat DRG and (2) to quantify the developmental changes in the mRNA expression of OCT2 in DRG neurons isolated from embryonic E15 and young adult Wistar rats. OCT2 mRNA expression in rat DRG was localized by in situ hybridization experiments and quantified by TaqMan Real Time PCR. Our results demonstrated the expression of OCT2 mRNA in rat DRG and its neuronal localization. In order to verify possible developmental changes in the OCT2 expression we quantified by TaqMan Real Time PCR the OCT2 mRNA using in vitro models of DRG neurons obtained from embryonic E15 rats and from young adult rats. OCT2 mRNA expression was lower in embryonic neuron cultures with respect to the one obtained from young adult rats, demonstrating a developmental change in the expression of this transporter in rat DRG neurons. The OCT2 mRNA expression in 4 weeks of age rats approached adult expression levels. Furthermore in literature is reported that the OCT2 mRNA expression in the kidney is gender dependent. Our preliminary data obtained in DRG of adult Wistar rats did not demonstrate the gender difference in OCT2 mRNA expression observed in the kidney. Key words: organic cation transporter, dorsal root ganglia neurons, developmental expression Supported in part by an unrestricted research grant from the “Fondazione Banca del Monte di Lombardia”

Italian Journal of Anatomy and Embryology, Vol 115, No 1/2 (Supplement) 2010

AIM.: The aim of this study is to evaluate the capability of Hyaloss™ matrix (Fab - Fidia Advanced Biopolymers - Pd - Italy), a biomaterial based on hyaluronic acid, used as organic scaffold in bone repair in post-extractive defects.20 post-extractive sockets were selected, with similar size defects in the same patient and in the same hemiarch. Hyaluronic acid with high molecular weight (Hyaloss™ matrix, Fab - Pd - Italy) was mixed with autologous bone obtained using Safescraper(®) curve (Meta - Re - Italy) to repair post-extractive sites. Safescraper(®) is a cutting edge system that allows to the collection of autologous bone without using traditional, incision-based collection techniques, which could cause discomfort to the patient.Clinical and hystological evaluations were performed, four months after grafting, in the maxilla and in the mandible. From a clinical point of view Hyaloss™ matrix mixed with autologous bone and patient's blood becomes a substance similar to gel, which is easy to insert in to the defect. From a hystological point of view, in the treated site there is the presence of an erosive activity, with accelerated angiogenetic and bone remodelling activities.The preliminary results show an acceleration of the bone deposit process and of its remodelling due to the presence of Hyaloss™ matrix, which, from a clinical point of view, improves the handling and application of the bone matrix inside the defects and, from a hystologic point of view makes it possible to obtain bone regeneration in less time when it is used with autologous bone.

Organic cation transporters (OCTs) are critical in drug absorption, targeting, and disposition. It has become increasingly clear the role of these transporters in key tissues responsible for drug absorption and disposition: kidney, liver and intestine. However, only limited information is available regarding their distribution and activity in nervous system. Cisplatin (CDDP), a widely used chemotherapeutic agent for the treatment of several solid tumors induces a severe and dose-limiting sensory neuropathy, due to damage of the primary sensory neurons of the dorsal root ganglia (DRG) where it forms DNA adducts. This study was aimed to investigate (1) the expression of different OCTs (OCT-1, OCT-2, OCTN-1 and OCTN-2) in DRG neurons and (2) their putative role in CDDP-induced peripheral neurotoxicity. OCTs expression in rat DRG was studied by TaqMan Real Time PCR in control and CDDP treated (2 mg/Kg i.p. 2qw x4) Wistar rats. It was demonstrated that CDDP administration reduced OCT2 mRNA levels with respect to the control. To examine the functional role of OCT-2, the correlation existing between the neurophysiologic/morphometric parameters and the OCT-2 mRNA levels were studied. This analysis evidenced that the reduction of OCT-2 expression correlated with the decrease of NCV and with the reduction of soma, nucleus and nucleolus size in treated animals. To obtain mechanistic information about the role of OCT-2 in CDDP neurotoxicity a pilot in vivo study was performed. Female Wistar rats were randomized in 3 different groups and treated as follows: CDDP 2 mg/Kg i.p. 2qw x4; CDDP 2 mg/Kg i.p. 2qw x4 + cimetidine (CMT) 60 mg/kg os daily for 4 weeks; un-treated control rats. The OCT-2 inhibitor CMT could not revert the CDDP-induced NVC impairment, although it significantly ameliorated morphometric parameters. An in vitro model of DRG neurons obtained from embryonic rats E15 was used.to confirm a partial neuroprotective effect of CMT. Nerve growth factor (NGF) differentiated neurons were exposed to CDDP alone or in combination with CMT. After 24 and 48 hours of co-treatment with CDDP and CMT 100 microM a significant increase of neuronal survival was observed with respect to CDDP treated cells. Our data suggest that rOCT2 could be involved in CDDP uptake in DRG neurons. The hypothesis is that CDDP treatment could determine a decrease in rOCT2 expression as a neuronal defense response against subsequent exposure and neuronal uptake of CDDP. Supported in part by an unrestricted research grant from the “Fondazione Banca del Monte di Lombardia”

Collagen is ubiquitous in the human body and it is the main component of connective tissue. Collagen is also the principal component of endopelvic fascia and it is involved in the physiopathology of female organ prolapse (POP). The role of collagen in the physiopathology of POP has been studied with conflicting results, being difficult to distinguish the cause from the effect and to standardize the samples. The turnover of connective tissue in the vaginal wall is an important process both in healthy women and in women with prolapse. MMP-1, MMP-8 and MMP-13 specifically cleave native triple collagen (I, II, III) helix, yelding two fragments. These fragments are subject to gelatinase (MMP-2 and MMP-9) degradation. MMP-2 and MMP-9 activity, may be regulated at different levels (trascriptional and activation level) influencing the amount of collagen. Aim of this study is to find a correlation between staging of anterior vaginal wall prolapse and morphologic and biochemical (MMP-2 and MMP-9 amount) features of anterior vaginal wall. Anterior vaginal wall full thickness biopsies (eightyfive patients) including endopelvic fascia have been collected during surgical procedures. Recurrence or vault prolapse have been ruled out. POP samples have been classified according to POP-Quantification and they have been divided in three groups: group A (controls), group B (I and II stage prolapse) and group C (III and IV stage prolapse). Each sample has been processed for both microscopic and zymography analysis. Formalin fixed samples have been embedded, cut and stained with Trichromic Masson Goldner (TMG) stains. Microscopic analysis have been performed to evaluate collagen amount, collagen organization, and to assess muscular component. All parameters have been scored semi-quantitatively by two blind observers on vaginal mucosa and underlying fascia. MMP-2 and MMP-9 activity have been evaluated by gelatine zymography. Statistical evaluation have been made by One-Way ANOVA analysis of variance. An increase in collagen content and a better connective organization in women with prolapse greater than III stage (group C p

Mesoporous silica particles (MSP) are a new development in nanotechnology. Covalent modification of the surface of the silica is possible both on the internal pore and on the external particle surface. It allows the design of functional nanostructured materials with properties of organic, biological and inorganic components. Research and development are ongoing on the MSP, which have applications in catalysis, drug delivery and imaging. The most recent and interesting advancements in size, morphology control and surface functionalization of MSP have enhanced the biocompatibility of these materials with high surface areas and pore volumes. In the last 5 years several reports have demonstrated that MSP can be efficiently internalized using in vitro and animal models. The functionalization of MSP with organic moieties or other nanostructures brings controlled release and molecular recognition capabilities to these mesoporous materials for drug/gene delivery and sensing applications, respectively. In this study different MSP have been tested: silica-FITC MSP and silica-folate-FITC MSP. Folic acid was used as the targeting ligand because -folate receptor is observed to be up-regulated in various types of human cancers. We have evaluated by confocal laser scanning microscopy (CLSM) the intracellular localization of FITC-loaded MSP in two different cancer cell lines: IGROV-1 (ovarian carcinoma) and A549 (lung adenocarcinoma) characterized by a different expression of folate receptor alpha. Our results demonstrate that silica nanoparticles localize into the cytoplasm of A549 and IGROV-1 cell lines after 1, 6 and 24 hours of treatment and present a perinuclear localization. Additionally the treated cells were examined cross-sectionally in order to confirm that the MSP were indeed internalized by the cells and not simply bound on the surface membrane. Flow cytometry was used for the quantification of fraction of cells that had internalized nanoparticles. The cellular uptake of silica nanoparticles was observed with both cell lines and folate modification on the silica nanoparticles increased the particles uptake by A549 and IGROV-1 cells. The over-expression of folate receptor may facilitate the recognition of the folate-modified nanoparticles and increase the uptake through folate receptor-mediated endocytosis Electron microscopy experiments also confirmed that silica nanoparticles are able to enter into tumor cells. The particles are progressively internalized into the cell cytoplasm starting from 15 minutes after treatment and after 30 minutes are completely internalized.

Organic cation transporters (OCTs) are critical in drug absorption, targeting, and disposition. It has become increasingly clear that multiple mechanisms are involved in organic cation transport in the key tissues responsible for drug absorption and disposition: the kidney, liver and intestine. However, only limited information is available regarding their distribution and activity in the central and peripheral nervous system. Cisplatin (CDDP), a widely used chemotherapeutic agent for the treatment of several solid tumors induces a severe and dose-limiting sensory neuropathy, due to damage of the primary sensory neurons of the dorsal root ganglia (DRG) where it forms DNA adducts. Previous in vivo and in vitro studies demonstrated that OCTs are implicated in CDDP nephrotoxicity. The present study was aimed to investigate (1) the presence of different OCTs (OCT-1, OCT-2, OCTN-1 and OCTN-2) in embryonic and adult rat DRG neurons and (2) their putative role in CDDP induced neurotoxicity. To examine the functional role of OCT-2, an in vivo study investigated the correlation existing between the neurophysiologic/morphometric parameters and the OCT-2 mRNA levels when rats were treated with CDDP. This analysis evidenced that the reduction of OCT-2 expression correlated with the decrease of NCV as well as with the reduction of soma, nucleus and nucleolus size in treated animals. Finally, to obtain mechanistic information about the role of OCT-2 in CDDP neurotoxicity we used an in vitro model of DRG neurons obtained from embryonic rats E15. The OCTs expression in rat DRG was studied by TaqMan Real Time PCR and it was demonstrated that CDDP administration reduced OCT-2 mRNA levels with respect to the control. In order to verify the involvement of OCT2 in the neuronal uptake of CDDP, nerve growth factor (NGF) differentiated neurons were exposed to CDDP alone or in combination with the OCT-2 inhibitor cimetidine (CMT). After 24 and 48 hours of co-treatment with CDDP and CMT 100 M we observed a significant increase of neuronal survival with respect to CDDP treated cells. Based on these results, a pilot in vivo study was performed that also confirmed a partial neuroprotective effect of CMT. Our data demonstrate that OCT-2 are present and are functionally active in rats. Moreover, it is likely that they are implicated in the pathogenesis of CDDP-induced DRG neuron damage, thus suggesting that they can be a target for future neuroprotective attempts. Supported in part by an unrestricted research grant from the Fondazione Banca del Monte di Lombardia

Chemotherapy induced Peripheral Neuropathy (CIPN) is a a well known complication limiting Cisplatin (CDDP) and Docetaxel (DOCE) chemotherapy in patients with cancer. Erythropoietin (EPO) is an heamatopoietic growth factor that has been reported to display neurothrophic properties, in particular it has proved to be neuroprotective in several rodent models of nervous system damage, including CDDP induced peripheral neuropathy. In this study we evaluated the neuroprotective ability of EPO against CDDP and DOCE induced neurotoxicity in primary cultures of sensitive neurons dissociated from Dorsal Root Ganglia (DRG) obtained from 15-day-old rat embryos, aiming to investigate the mechanism underlying EPO neuroprotective action. Briefly, primary cultures of sensitive neurons have been exposed to CDDP (25M), DOCE (100nM) or to the same doses of the chemotherapics but in the presence of EPO (10nM). In this in vitro model EPO turned out to be protective against both CDDP and DOCE induced neurotoxicity, in fact an increased neuronal survival was evident in cultures exposed to the combination treatment with CDDP plus EPO and DOCE plus EPO. Several evidences suggest that AKT pathway may play an important role in EPO neuroprotective action. Therefore we analyzed by western blot the levels of expression and activation (phosphorylation) of AKT in neurons exposed to the CDDP, DOCE or to the cotreatment with EPO. Results obtained indicated that modulation of the levels of phosphorylated AKT (pAKT) occurs following 14 hours treatment with CDDP, when a sharp decrease in the levels of activated AKT was highlighted, on the contrary DOCE induced only a sligth increase in the level of pAKT. Cotreatment with EPO was able to partially prevent CDDP induced decrease in the level of pAKT. Using a specific inhibitor of AKT activation (Wortmannin) we therefore investigate the role of AKT in EPO neuroprotection. The presence of Wortmannin (100nM) did prevent EPO neuroprotective action against CDDP induced neurotoxicity, but did not have any effect on EPO action against DOCE induced neurotoxicity. In conclusion in this study EPO has proved to be an effective neuroprotectant against neurotoxicity induced by both Cisplatin and Docetaxel, in addition results obtained suggest that AKT is an essential element in EPO neuroprotection. However, considering that AKT activation appears essential for EPO neuroprotection against CDDP induced neurotoxity, but not against DOCE, we assume that EPO neuroprotective action may act on two distinct cellular signalling pathways following CDDP or DOCE induced neurotoxicity.

Several antineoplastic drugs induce severe toxic damage of the peripheral nervous system and chemotherapy-induced peripheral neurotoxicity (CIPN) can be dose limiting. Moreover, CIPN signs and symptoms can be permanent and severely impair the patients' quality of life even after drug withdrawal. Despite extensive investigation, the exact mechanisms of neurotoxic action at the basis of CIPN are not completely known and it is likely that they can be at least in part different from the mechanisms of antineoplastic action of the drugs. A possible instrument to investigate on this important issue is represented by the evaluation of the effect of compounds used to reduce the toxicity of antineoplastic drugs in pre-clinical and clinical settings. This review will be focused on the most clinically-relevant neurotoxic antineoplastic drugs and on the results obtained with several different classes of putative neuroprotectants.

Collagen is ubiquitous in the human body and it is the main component of connective tissue. Collagen is also the main component of the pelvic supporting structures and Pelvic Organ Prolapse (POP) is the clinical manifestation of a muscular and connective tissue damage. The role of collagen in the physiopathology of POP has been studied with conflicting results, being difficult to distinguish the cause from the effect and to standardize the samples. In this study it has been evaluated the microscopic morphology of the anterior and posterior vaginal wall of patients affected by different stages of POP. Vaginal wall biopsies have been obtained during surgical procedures performed from January 2007 to January 2008. Each biopsy of anterior vaginal wall was provided during cystocele repair as a midline longitudinal full thickness excision including pelvic fascia. Biopsies on posterior compartment were obtained during posterior repair procedures also including a longitudinal midline excision of the vaginal wall and rectovaginal space. Posterior biopsies were further divided according to POP-Q classification system in Ap, Bp, and D point respectively. All samples have been grouped according to the ICS prolapse stadiation. Recurrences or vault prolapses have been ruled out. Microscopic evaluation on Hematossilin & Eosin (H&E) stained sections was performed to quantify inflammatory infiltration and vascularization. Microscopic evaluation on Trichromatic Masson Goldner (TMG) stained sections was performed to assess collagen amount, organization and composition and to assess the muscular component. All parameters were scored semi-quantitatively by two blind observers on two areas of interest. Results show statistically significant difference of collagen amount among different stages of prolapse. Statistically significant differences are also evident in the posterior compartment considering the amount of muscular tissue. Ceresa C is supported by “Fondazione Policlinico di Monza”

The experimentally induced neurotoxic effects of paclitaxel and docetaxel have never been compared, since no animal models of docetaxel peripheral neurotoxicity have yet been reported. In this experiment, we examined the effect of the chronic administration of these two taxanes in the Wistar rat using neurophysiological, neuropathological and morphometrical methods. Our results showed that both paclitaxel and docetaxel induced a significant, equally severe and dose-dependent reduction in nerve conduction velocity. On the contrary, the morphometric examination demonstrated that the effect on the nerve fibres was more severe after paclitaxel administration when the same schedule was used. However, the overall severity of the pathological changes was milder than expected on the basis of the neurophysiological results. Our results support the hypothesis that taxanes (and particularly docetaxel) may exert their neurotoxic effect not only on the microtubular system of the peripheral nerves, but also on other less obvious targets.