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The insurgence of drug resistance in treating Multiple Myeloma (MM) still represents a major hamper in finding effective treatments, although over the past decades new classes of drugs, such as proteasome inhibitors and immunomodulatory drugs, have been discovered. Recently, our research team, within a Nature-Aided Drug Discovery project, isolated from Hibiscus Sabdariffa L. calyces the secondary metabolite called Hib-ester which possesses antiproliferative properties against human multiple myeloma RPMI 8226 cells, reduces migration and cell invasion and inhibits proteasome without neurotoxic effects. In the present study, we explored the chemical spaces of the hit compound Hib-ester. We explored the structure-activity relationships (SAR), and we optimized the scaffold through sequentially modifying Hib-ester subunits. Compound screening was performed based on cytotoxicity against the RPMI 8226 cells to assess the potential efficacy toward human MM. The ability of the most effective molecules to inhibit the proteasome was evaluated and the binding mode of the most promising compounds in the proteasome chymotrypsin binding pocket was deciphered through molecular modeling simulations. Compounds 13 and 14 are more potent than Hib-ester, demonstrating that our strategy was suitable for the identification of a novel chemotype for developing possible drug candidates and hopefully widening the drug armamentarium against MM.

Hibiscus Sabdariffa (HS) is a plant of the Malvacee family commonly cultured in tropical and subtropical countries. It is mainly known as the main ingredient for the preparation of cold drink called Karkadè. Calices and leaves of HS plant are also used in folk medicine thanks to their antioxidant and anti-inflammatory properties. In recent years, HS has also gained great interest as a possible antitumoral agent. In the present PhD project, we evaluated the antitumoral effects of HS against multiple mye-loma cells in vitro. Multiple myeloma is the most frequent hematological malignancy world-wide. In recent years, new drugs have increased the survival expectancy of patients. Despite this, new therapeutic approaches are necessary, especially for high multiple myeloma hetero-geneity and for relapsed or refractory multiple myeloma. The project was organized in three distinct phases: 1- Evaluation of antitumoral effects of HS against RPMI 8226 human multiple myeloma cells. We demonstrated by MTT and Trypan blue assays that a total HS extract (HSE) and one of its fraction obtained by liquid-liquid extraction (HSEC) were able to impair cell viability of human multiple myeloma RPMI 8226 in a dose and time dependent manner. HSE cell viability reduction was due to a cytostatic action, while HSEC was more cytotoxic and induced a caspase dependent apoptosis. Moreover, both HSE and HSEC impaired cell migration and invasion of RPMI 8226 cells in a Boyden chamber as-say. We also demonstrated in in vitro model of neurotoxicity (dorsal root ganglia model) that HSE and HSEC concentrations used in our experiments were not neurotoxic. In RPMI 8226 cells autophagy and proteasome activity were impaired by both HSE and HSEC. MAPK p38 activation was observed in the first 6h of treatment, while ERK 1 and ERK 2 activation occurred between 16 and 48h. 2- Evaluation of combinations between Bortezomib (BTZ) and HSE or HSEC against RPMI 8226 multiple myeloma cells. We evaluated several combinat, Hibiscus Sabdariffa (HS) is a plant of the Malvacee family commonly cultured in tropical and subtropical countries. It is mainly known as the main ingredient for the preparation of cold drink called Karkadè. Calices and leaves of HS plant are also used in folk medicine thanks to their antioxidant and anti-inflammatory properties. In recent years, HS has also gained great interest as a possible antitumoral agent. In the present PhD project, we evaluated the antitumoral effects of HS against multiple mye-loma cells in vitro. Multiple myeloma is the most frequent hematological malignancy world-wide. In recent years, new drugs have increased the survival expectancy of patients. Despite this, new therapeutic approaches are necessary, especially for high multiple myeloma hetero-geneity and for relapsed or refractory multiple myeloma. The project was organized in three distinct phases: 1- Evaluation of antitumoral effects of HS against RPMI 8226 human multiple myeloma cells. We demonstrated by MTT and Trypan blue assays that a total HS extract (HSE) and one of its fraction obtained by liquid-liquid extraction (HSEC) were able to impair cell viability of human multiple myeloma RPMI 8226 in a dose and time dependent manner. HSE cell viability reduction was due to a cytostatic action, while HSEC was more cytotoxic and induced a caspase dependent apoptosis. Moreover, both HSE and HSEC impaired cell migration and invasion of RPMI 8226 cells in a Boyden chamber as-say. We also demonstrated in in vitro model of neurotoxicity (dorsal root ganglia model) that HSE and HSEC concentrations used in our experiments were not neurotoxic. In RPMI 8226 cells autophagy and proteasome activity were impaired by both HSE and HSEC. MAPK p38 activation was observed in the first 6h of treatment, while ERK 1 and ERK 2 activation occurred between 16 and 48h. 2- Evaluation of combinations between Bortezomib (BTZ) and HSE or HSEC against RPMI 8226 multiple myeloma cells. We evaluated several combinat

Human bone marrow mesenchymal stem cells (hBM-MSCs) are the best characterized multipotent adult stem cells. Their self-renewal capacity, multilineage differentiation potential, and immunomodulatory properties have indicated that they can be used in many clinical therapies. In a previous work we studied the DNA methylation levels of hBM-MSC genomic DNA in order to delineate a kind of methylation signature specific for early and late passages of culture. In the present work we focused on the modification of the methylation profiles of the X chromosome and imprinted loci, as sites expected to be more stable than whole genome. We propose a model where cultured hBM-MSCs undergo random modifications at the methylation level of most CGIs, nevertheless reflecting the original methylation status. We also pointed out global genome-wide demethylation connected to the long-term culture and senescence. Modification at CGIs promoters of specific genes could be related to the decrease in adipogenic differentiation potential. In conclusion, we showed important changes in CGIs methylation due to long-term in vitro culture that may affect the differentiation potential of hBM-MSCs. Therefore it is necessary to optimize the experimental conditions for in vitro expansion in order to minimize these epigenetic changes and to standardize safer procedures.

Cancer is a leading cause of death worldwide. Despite therapeutic improvements, some cancers are still untreatable. Recently there has been an increasing interest in the use of natural substances for cancer prevention and treatment. Hibiscus sabdariffa (HS) is a plant, belonging to Malvaceae family, widespread in South Asia and Central Africa. HS extract (HSE) used in folk medicine, gained researchers' interest thanks to its antioxidant, anti-inflammatory, and chemopreventive properties. In the present study, we initially assessed HSE effect on a panel of human tumor cell lines. Then we focused our study on the following that are most sensitive to HSE action cell lines: Multiple Myeloma (MM) cells (RPMI 8226) and Oral Squamous Cell Carcinoma (OSCC) cells (SCC-25). In both RPMI 8226 and SCC-25 cells, HSE impaired cell growth, exerted a reversible cytostatic effect, and reduced cell motility and invasiveness. We evaluated the involvement of MAPKs ERK1/2 and p38 in HSE effects by using specific inhibitors, U0126 and SB203580, respectively. For both SCC-25 and RPMI 8226, HSE cytostatic effect depends on p38 activation, whereas ERK1/2 modulation is crucial for cell motility and invasiveness. Our results suggest that HSE may be a potential therapeutic agent against MM and OSCC.

Background/Aim: Peripheral neurotoxicity is a dose-limiting factor of many chemotherapeutic agents, including cisplatin. Mesenchymal stem cells are promising for the treatment of several neurological disorders, and our aim was to verify the neuroprotective potential of human mesenchymal stem cells (hMSCs) on dorsal root ganglia (DRG) exposed to cisplatin. Materials and Methods: DRG were exposed to different cisplatin concentrations and then co-cultured with hMSCs or with hMSC-conditioned medium. Results: hMSCs showed a neuroprotective effect on cisplatininduced death of DRG, mediated by direct contact. Moreover, DRG exhibited an MSC-dependent promotion of neurite outgrowth, in particular at early time points. For this effect, the expression of Neurite Outgrowth Inhibitor (NOGO) and Myelin Associated Glycoprotein (MAG) by hMSCs was pivotal. Conclusion: hMSCs are a promising tool for reducing the neurotoxic effect of cisplatin.

Background and Objectives: Cellular therapies using Mesenchymal Stem Cells (MSCs) represent a promising approach for the treatment of degenerative diseases, in particular for mesengenic tissue regeneration. However, before the approval of clinical trials in humans, in vitro studies must be performed aimed at investigating MSCs' biology and the mechanisms regulating their proliferation and differentiation abilities. Besides studies on human MSCs (hMSCs), MSCs derived from rodents have been the most used cellular type for in vitro studies. Nevertheless, the transfer of the results obtained using animal MSCs to hMSCs has been hindered by the limited knowledge regarding the similarities existing between cells of different origins. Aim of this paper is to highlight similarities and differences and to clarify the sometimes reported different results obtained using these cells. Methods and Results: We compare the differentiation ability into mesengenic lineages of rat and human MSCs cultured in their standard conditions. Our results describe in which way the source from which MSCs are derived affects their differentiation potential, depending on the mesengenic lineage considered. For osteogenic and chondrogenic lineages, the main difference between human and rat MSCs is represented by differentiation time, while for adipogenesis hMSCs have a greater differentiation potential. Conclusions: These results on the one hand suggest to carefully evaluate the transfer of results obtained with animal MSCs, on the other hand they offer a clue to better apply MSCs into clinical practice

Cholangiocarcinoma (CCA) is the second most frequent primary liver neoplasia. It mainly arises from the malignant transformation of biliary epithelial cells, although it might originate from either hepatic progenitor cells at the Hering canals or transformed hepatocytes. CCA is a highly aggressive tumor with extremely poor prognosis and limited therapeutic approaches. Baicalin (BA) is one of the main bioactive flavonoids identified in the Scutellaria Baicalensis Georgi root dried extract which is extensively used in the Chinese tra-ditional medicine. Together with the anti-inflammatory effect, the anti-neoplastic action is the most relevant BA property demonstrated on cancer cells of different origin. Being aware of the need of new therapeutic weapons for CCA treatment, we in-vestigated whether Baicalin could exert anti-proliferative and anti-migratory effect on EGI-1 cells, a highly metastatic CCA cell line derived from bile duct carcinoma. We first tested different BA concentrations (from 5 to 200µM) in limiting EGI-1 via-bility using MTT assay. After 24h and 48h treatment, 5 and 10µM BA had no effect while rising from 25µM to 200µM (i.e. 25,50,100 and 200µM) BA exerted a significant cell viability reduction already at 24h and increased after 48h BA expo-sure. This reduction well correlated with the adherent absolute cell number de-crease and it cannot be due to BA induced cell cycle impairment after neither 24 nor 48h treatment. We also evaluated the anti-migratory BA potential by a wound healing assay adding different BA concentrations (5, 25, 50,100 and 200µM) to the culture medium immediately after performing a wound on confluent cell cultures. All BA concen-trations but 5µM induced a significant reduction in the EGI-1 migration rate after 24h treatment. Moreover 25, 50 and 10µM BA showed similar migration inhibition extent at 24 and 48h whilst 200µM BA exerted a stronger inhibitory effect already after 24h exposure which increased with time in a significant w

RIASSUNTO E SCOPO DEL LAVORO Esiste una stretta correlazione tra obesità e osteoporosi: nell'obesità si riscontra una maggiore fragilità ossea ed un ridotto assorbimento di calcio a livello intestinale, mentre l'osteoporosi è spesso accompagnata da un aumento dell'adipogenesi midollare. In questi ultimi anni un'area della ricerca scientifica si è concentrata sullo studio di nuovi farmaci/sostanze in grado di agire sull'adipogenesi o sull'osteoblastogenesi in quanto è fondamentale trovare nuove terapie che prevengano tali patologie. Conoscendo la stretta relazione tra il differenziamento adipogenico e osteogenico, un farmaco contro l'obesità non dovrebbe avere effetti sul metabolismo osseo, mentre contro l'osteoporosi non dovrebbe avere effetti sull'adipogenesi. A tale proposito gli studi in vitro rappresentano un punto importante sia per valutare l’effetto di tali sostanze che per comprendere i meccanismi alla base delle loro azioni. La maggior parte degli studi condotti sul differenziamento adipocitico, e sulla sua inibizione, sono stati condotti su linee di preadipociti murini (3T3-L1) o umani, mentre sono state utilizzate linee murine MC3T3-E1 e C3H10T1/2 per il differenziamento osteogenico. Nonostante la rilevanza dei dati ottenuti grazie a questi modelli cellulari, i risultati sono spesso controversi, anche in considerazione del fatto che i processi differenziativi nell’uomo e nei roditori possono essere regolati a livello molecolare in modo significativamente diverso. Inoltre questi modelli sono basati su cellule già indirizzate verso il differenziamento adipocitico o osteogenico, e quindi non prendono in considerazione il processo di determinazione che porta la cellula progenitrice ed indifferenziata verso un lineage mesengenico. Le cellule staminali mesenchimali (MSC) in questo contesto rappresentano un modello cellulare particolarmente utile in quanto possono essere efficacemente indotte da uno stato indifferenziato allo stato di adipociti o osteobl

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 re-duction 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 ex-tract 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 re-spectively, 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 i

Bortezomib (BZ), a proteasome inhibitor, is an antineoplastic drug used in the treatment of multiple myeloma and mantle cell lymphoma. Its main clinical and dose-limiting side effect is the development of painful peripheral neuropathy. Despite BZ-induced peripheral neuropathy (BiPN) substantially reduces patients quality of life, no effective therapies have been development so far. Here we worked up in vitro and in vivo models to deeper investigate the pathogenesis of BiPN. Since BZ alters protein degradation, we supposed that proteins regulating microtubule (MT) stability may be altered after treatment. Moreover, since sensory neurons have a primary role in the development of BiPN, we treated cultured DRG sensory neurons with BZ (100nM) for 48h and 72h, and we evaluated tubulin polimerization by comparing the distribution of tubulin in polymerized (P) and soluble (S) fractions. The increase of MT polymerization following treatment of DRG sensory neurons was observed; the proportion of tubulin in polymerized fraction was increased after 48h and 72 h of BZ-treatment. To verify this mechanism also in an in vivo model, we examined MT polymerization on both acute and chronic model of BiPN. In the acute model a single-dose of 0.20 mg/kg of BZ was injected in Wistar rat, and then the polimerization was analysed in sciatic nerves after 48h and 72h. In the chronic model animals were treated with BZ 0.20 mg/kg, three times/week for eight weeks, and the neurophysiological and neuropathological damages were observed in peripheral nerves. At the end of the treatment and after 2 weeks of follow-up period, the MT polymerization in sciatic nerve was measured by western blot. In the acute schedule of treatment, the increase of tubulin in polymerized fraction was observed within 72 hours from BZ administration. Similar result was observed also at the end of chronic BZ-administration. The level of tubulin polimerization was returned to physiological levels at the end of the follow-up

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

Mesenchymal stemcells (MSCs) hold great promise for the treatment of numerous diseases. A major problem for MSC therapeutic use is represented by the very low amount of MSCs which can be isolated from different tissues; thus ex vivo expansion is indispensable. Long-term culture, however, is associated with extensive morphological and functional changes of MSCs. In addition, the concern that they may accumulate stochastic mutations which lead the risk of malignant transformation still remains. Overall, the genome of human MSCs (hMSCs) appears to be apparently stable throughout culture, though transient clonal aneuploidies have been detected. Particular attention should be given to the use of low-oxygen environment in order to increase the proliferative capacity of hMSCs, since data on the effect of hypoxic culture conditions on genomic stability are few and contradictory. Furthermore, specific and reproducible epigenetic changes were acquired by hMSCs during ex vivo expansion, which may be connected and trigger all the biological changes observed. In this review we address current issues on long-term culture of hMSCs with a 360-degree view, starting from the genomic profiles and back, looking for an epigenetic interpretation of their genetic stability

The spontaneous expression of neural markers by mesenchymal stem cells (MSCs) has been considered to be a demonstration of MSCs' predisposition to differentiate towards neural lineages. In view of their application in cell therapy for neurodegenerative diseases, it is very important to deepen the knowledge about this distinctive biological property of MSCs. In this study, we evaluated the expression of neuronal and glial markers in undifferentiated rat MSCs (rMSCs) at different culture passages (from early to late). rMSCs spontaneously expressed neural markers depending on culture passage, and they were coexpressed or not with the neural progenitor marker nestin. In contrast, the number of rMSCs expressing mesengenic differentiation markers was very low or even completely absent. Moreover, rMSCs at late culture passages were not senescent cells and maintained the MSC immunophenotype. However, their differentiation capabilities were altered. In conclusion, our results support the concept of MSCs as multidifferentiated cells and suggest the existence of immature and mature neurally fated rMSC subpopulations. A possible correlation between specific MSC subpopulations and specific neural lineages could optimize the use of MSCs in cell transplantation therapy for the treatment of neurological diseases. © 2012 Dana Foudah et al.

Intracellular transport is fundamental for neuronal function and survival. The majority of proteins are synthesized in the neuron cell body and transported along axons and dendrites through molecular motors as the Kinesin superfamily proteins (KIFs). Two specific KIFs that have been associated strongly with neurodegenerative processes in humans and in rodents are KIF5A and KIF21B. In fact, KIF5A down regulation has been associated with axonal transport defects in models of multiple sclerosis (MS) and a genome wide association screen for MS correlated single nucleotide polymorphisms located in the KIF21B intron with the disease, establishing this kinesin as a susceptibility locus for MS. Since nitric oxide (NO) has a key role in mediating inflammatory axonopathy in MS promoting protein mis-folding, disruption of mitochondrial respiratory chain and organelle fragmentation, the first aim of the present study was to determine the effect of NO exposure on the expression of KIF5A and KIF21B in rodent cortical neurons and to evaluate whether KIFs expression correlates with axon pathology. Results demonstrated that NO cause a time dependent decrease of gene and protein expression for both KIF proteins. Furthermore, dot blot analysis showed that NO cause a time dependent decrease in axon phosphorylation and that KIFs reduction precede the loss of neurofilament. Human bone marrow mesenchymal stem cells (MSCs) represent a promising candidate for neuronal repair due to anti-inflammatory, antioxidant and neurotrophic properties. The second part of this study was therefore to investigate the capacity of MSC to protect neurons and axonal transport mechanisms in rodent cortical neurons exposed to NO. Results showed that MSC were able to preserve axonal length and increase survival in cortical neurons exposed to NO, furthermore MSCs had the ability to preserve both KIF5A and KIF21B protein expression from nitric oxide damage. Finally in this study, it was evaluated if there were any

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 Ano

The induction of a peripheral neuropathy is a very common side effect of many chemotherapeutic agents, including platinum compounds, and it often represents the dose limiting factor for drug clinical use. Several strategies have been suggested to reduce drug neurotoxicity without affecting the antineoplastic potential, but up to now results were not encouraging. Recently, it has been demonstrated that Mesenchymal Stem Cells (MSCs) are able to promote the survival and the maturation of untreated sensory neurons of dorsal root ganglia (DRG), which represent also the target of drug neurotoxicity. Aim of this work is to verify the neuroprotective potential of MSCs on rat DRG exposed to cisplatin (CDDP), a chemotherapeutic and neurotoxic agent. DRG post-mitotic explants from E15 rat embryos were exposed for 24 hours to different cisplatin concentrations. After 24 hours, medium was changed and DRG were directly co-cultured with human MSCs (hMSCs) or with hMSCs conditioned medium (hMSC-CM). DRG explants were photographed every day up to 1 month, and the longest neurite of each DRG was measured to evaluate neurotoxicity. DRG survival was estimated by measuring the death area percentage. The survival of CDDP-treated DRG was increased after the co-cultures with hMSCs, and both hMSCs and hMSC-CM were able to improve the neurite outgrowth of untreated and CDDP-treated DRG after 48 hours. This MSC-dependent increase of neurite length was however no longer evident at later times (1 month). This effect on neurite elongation was probably mediated by CSPG, MAG and Nogo, some proteins involved in the modulation of neurite elongation, which resulted expressed and released in the culture medium of hMSCS. Our results demonstrated a neuroprotective effect of hMSCs on CDDP toxicity and evidenced the ability of these cells to modulate neurite elongation. In this way MSCs could represent a possible mean to limit the neurotoxicity on DRG which arises after cisplatin therapy.

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

Introduction. Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent cells that can differentiate into different cell lineages and have emerged as a promising tool for cell-targeted therapies and tissue engineering. Their use in a therapeutic context requires large-scale in vitro expansion, increasing the probability of genetic and epigenetic instabilities. Some evidence shows that an organized program of replicative senescence is triggered in human BM-MSCs (hBM-MSCs) on prolonged in vitro expansion that includes alterations in phenotype, differentiation potential, telomere length, proliferation rates, global gene-expression patterns, and DNA methylation profiles. Methods. In this study, we monitored the chromosomal status, the biologic behavior, and the senescence state of hBM-MSCs derived from eight healthy donors at different passages during in vitro propagation. For a more complete picture, the telomere length was also monitored in five of eight donors, whereas the genomic profile was evaluated in three of eight donors by array-comparative genomic hybridization (array-CGH). Finally, an epigenomic profile was delineated and compared between early and late passages, by pooling DNA of hBM-MSCs from four donors. Results: Our data indicate that long-term culture severely affects the characteristics of hBM-MSCs. All the observed changes (that is, enlarged morphology, decreased number of cell divisions, random loss of genomic regions, telomere shortening) might be regulated by epigenetic modifications. Gene Ontology analysis revealed that specific biologic processes of hBM-MSCs are affected by variations in DNA methylation from early to late passages. Conclusions: Because we revealed a significant decrease in DNA methylation levels in hBM-MSCs during long-term culture, it is very important to unravel how these modifications can influence the biologic features of hBM-MSCs to keep track of this organized program and also to clarify the conflicting observations on h

Multiple sclerosis (MS) is a chronic immuno-mediated inflammatory and demyelinating disease characterised by the presence of both demyelinating lesions and axonal degeneration, which lead to the reduction of nerve conduction velocity and the development of concomitant neurological deficits. Recently, Mesenchymal Stem Cells (MSCs) have been proposed in in vivo studies as promising therapeutic treatment for MS mainly for their capacity to modulate the immune response, moreover, during our previous experiments we found that rat undifferentiated MSC promote Dorsal Root Ganglia neurons survival and maturation. The aim of this study is to verify the potential protective effect of MSCs on an in vitro model of MS represented by rat primary cultures of cortical neurons. Since glutamate excitotoxicity is an important mechanism in neurodegenerative diseases, and it induces neuronal alterations similar to those observed in advanced MS, cortical neurons cultures were treated with different concentrations of glutamate (25, 50, 100, 200 and 500 µM) for 24 hours. After the treatment cortical neurons show a suffering appearance, with damaged axons and cellular degeneration. Neuronal viability, assessed by DAPI staining and by count of viable cells, was glutamate dose-dependent. In order to evaluate the possible positive effect of MSCs on neuronal survival, both direct and indirect co-cultures of MSCs and cortical neurons were set up, and the survival after glutamate treatment analyzed. Cortical neurons treated with glutamate were still suffering after the direct co-cultures with MSCs, while in indirect co-cultures with MSCs neurons were alive, without important signs of axonal degeneration. These preliminary findings suggest that MSCs are able to reduce the cellular death induced by glutamate exposure in cortical neurons and encouraged the further study and characterization of their positive effect

Mesenchymal Stem Cells (MSCs) are a bone marrow-derived population present in adult tissues that possess the important property of dividing when called upon and of differentiating into specialized cells. The evidence that MSCs were able to transdifferentiate into specialized cells of tissues different from bone marrow, in particular into nervous cells, opened up the possibility of using MSCs to substitute damaged neurons, that are normally not replaced but lost, in order to repair the Nervous System. The first neuronal differentiation protocols were based on the use of a mixture of toxic drugs which induced MSCs to rapidly acquire a neuronal-like morphology with the expression of specific neuronal markers. However, many subsequent studies demonstrated that the morphological and molecular modifications of MSCs were probably due to a stress response, rather than to a real differentiation into neuronal cells, thus throwing into question the possible use of MSCs to repair the nervous system. Currently, some papers are suggesting again that it may be possible to induce neuronal differentiation of MSCs by using several differentiation protocols, and by accompanying the morphological evidence of differentiation with functional evidence, thus demonstrating that MSC-derived cells not only seem to be neurons, but that they also function like neurons. In this review, we have attempted to shed light on the capacity of MSCs to genuinely differentiate into nervous cells, and to identify the most reliable protocols for obtaining neurons from MSCs for nervous system repair

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.

ntroduction. Mesenchymal Stem Cells (MSCs) are adult multipotent cells able to differentiate in mesengenic (osteogenic, adipogenic, condrogenic) and non mesengenic lineages (e.g. neural) under appropriate culture conditions. MSCs represent a very promising therapeutic approach in different settings particularly for tissue repair and regeneration. The knowledge of human MSCs (hMSCs) biological properties is very important to optimize their clinical application. In view of MSCs application in neurodegenerative diseases, the neuronal differentiation potential of hMSCs has been also explored. Our preliminary data demonstrated that the neuronal markers beta III tubulin and NeuN were spontaneously expressed by a high percentage of undifferentiated hMSCs independently from serum presence and number of culture passages. The expression of neural markers by MSCs in absence of any differentiative agents is considered as a demonstration of MSC neural predisposition. The aim of this work was to evaluate if these markers, known to be neuronal ones, continued to be expressed also in hMSCs differentiated towards mesengenic lineages. Methods. hMSCs were obtained after patient consensus, from iliac crest bone marrow. In according to the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy, the isolated hMSCs were plastic-adherent, capable of extensive proliferation when maintained in standard culture conditions, lacked of hematopoietic markers expression and presented specific surface antigens. hMSCs were differentiated toward osteogenic, adipogenic and chondrogenic lineages using specific in vitro protocols. The expression of the neuronal markers beta III tubulin and NeuN were evaluated by immunofluorescence experiments at different time points depending on the differentiation protocol used. hMSCs cultured in absence of any differentiative agent represented controls. Results. In our experiments the most of hMSCs differentiated in osteogenic an

Adipocytes' biology and the mechanisms that control adipogenesis have gained importance because of the need to develop therapeutic strategies to control obesity and the related pathologies. Human mesenchymal stem cells (hMSCs), undifferentiated stem cells present in the bone marrow that are physiological precursors of adipocytes, were induced to adipogenic differentiation. The molecular mechanisms on the basis of the adipogenesis were evaluated, focusing on the MAPKinases ERK1 and ERK2, which are involved in many biological and cellular processes. ERK1 and ERK2 phosphorylation was reduced with different timing and intensity for the two isoforms in treated hMSCs in comparison with control cells until day 10 and then at 14-28 days, it reached the level of untreated cultures. The total amount of ERK1 was also decreased up to day 10 and then was induced to the level of untreated cultures, whereas the expression of ERK2 was not changed following adipogenic induction. Treatment with the specific ERK1/2 inhibitor U0126 during the whole differentiation period hampered hMSCs' adipogenic differentiation, as lipid droplets appeared in very few cells and were reduced in number and size. When U0126 was administered only during the initial phase of differentiation, the number of hMSCs recruited to adipogenesis was reduced while, when it was administered later, hMSCs did not acquire a mature adipocytic phenotype. ERK1 and ERK2 are important for hMSC adipogenic differentiation since any alteration to the correct timing of their phosphorylation affects either the recruitment into the differentiation program and the extent of their maturation.

Type 1 diabetes is a chronic disease often leading to systemic complications, such as peripheral neuropathy, nephropathy and cardiovascular complications. Whole pancreas as well as pancreatic islets transplantation has been proposed for the cure of type 1 diabetes, allowing a more efficient and physiological metabolic control. We investigated the effects of microencapsulated and immunoisolated islet transplantation in a model of streptozotocin-induced diabetes in 3 groups of Lewis rats: healthy controls, untreated diabetic rats and diabetic rats transplanted with microencapsulated islets into the peritoneal cavity two months after diabetes induction. Our results demonstrated that following transplantation hyperglycemic rats became normoglycemic in few days and this was accompanied by a rapid raise in body weight. Meanwhile, thermal (hot plate test) and mechanical sensitivity (Randal-Selitto paw withdrawal test measured with an analgesymeter) were increased and decreased by 180 and 40-60%, respectively. In addition, the density of footpad intraepidermal nerve fibers was significantly reduced by 20% in diabetic group and islet transplantation restored normal skin innervation. Nerve conduction velocity in the tail nerve and the Na+, K+-ATPase activity in the sciatic nerve, both reduced by about 25% in diabetic rats, were also normalized by islet transplantation. In conclusion, our data obtained in a model of type 1 diabetes, showed that transplant of microencapsulated pancreatic islets, besides controlling glycemia, arrested neuropathy worsening and was able to restore all the diabetic-induced alterations within the 2-month follow-up period after transplantation. On the basis of these encouraging results, a new experiment with 4-month long diabetes followed-up for 4 months after allogenic transplantation has been performed and the preliminary analysis confirmed the effectiveness of the procedure also in these long-term diabetic animals. In fact, effective treatment of

Aims In order to develop therapeutic strategies to control obesity and the related pathologies, it is very important to deepen the knowledge of adipocytes biology and the mechanisms that control adipogenesis. In the present work we have studied the molecular mechanisms at the basis of the adipocytes differentiation using the human Mesenchymal Stem Cells (hMSCs), undifferentiated stem cells present in the bone marrow that are the physiological precursors of adipocytes in the organism. In particular we have focused our attention on the MAPKinases ERK1 and ERK2, that are involved in many biological and cellular processes. Methods hMSCs, obtained from iliac crest bone marrow, were induced to adipogenic differentiation by treatment with Adipogenic Induction Medium for 10 days (determination phase), then replaced with Adipogenic Maintenance Medium until the end of treatment at day 28 (terminal differentiation phase). hMSC adipogenic differentiation was evaluated by morphological and molecular techniques. Control cells were represented by hMSCs cultured in absence of adipogenic supplements. ERK1 and ERK2 expression and phosphorylation were evaluated by immunoblotting experiments. The specific ERK inhibitor U0126 was added to the adipogenic medium at different times during the adipogenic differentiation protocol. Results hMSC treated with adipogenic differentiation protocol showed lipid droplets that increased in number and size during the whole differentiation period. In treated hMSC both ERK1 and ERK2 phosphorylation was reduced in comparison to control hMSCs, but time and intensity of these reductions were different for the two isoforms. A decrease of the total amount of ERK1 was also observed. The presence of U0126 during the whole differentiation period hampered the adipogenic differentiation of hMSCs, as very few hMSCs showed the appearance of lipid droplets that were reduced both in number and size. When U0126 was administered only during the determination phase the

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