Extracellular Matrix Structure and Interaction with Immune Cells in Adult Astrocytic Tumors.

The extracellular matrix (ECM) is a dynamic set of molecules produced by the cellular component of normal and pathological tissues of the embryo and adult. ECM acts as critical regulator in various biological processes such as differentiation, cell proliferation, angiogenesis, and immune control. The most frequent primary brain tumors are gliomas and by far the majority are adult astrocytic tumors (AATs). The prognosis for patients with these neoplasms is poor and the treatments modestly improves survival. In the literature, there is a fair number of studies concerning the composition of the ECM in AATs, while the number of studies relating the composition of the ECM with the immune regulation is smaller. Circulating ECM proteins have emerged as a promising biomarker that reflect the general immune landscape of tumor microenvironment and may represent a useful tool in assessing disease activity. Given the importance it can have for therapeutic and prognostic purposes, the aim of our study is to summarize the biological properties of ECM components and their effects on the tumor microenvironment and to provide an overview of the interactions between major ECM proteins and immune cells in AATs. As the field of immunotherapy in glioma is quickly expanding, we retain that current data together with future studies on ECM organization and functions in glioma will provide important insights into the tuning of immunotherapeutic approaches.

Food Safety Assessment and Nutraceutical Outcomes of Dairy By-Products: Ovine Milk Whey as Wound Repair Enhancer on Injured Human Primary Gingival Fibroblasts.

The valorization of milk whey appears to be a promising strategy for managing by-products from dairy food industries, which incur demanding economic costs for treatment and/or disposal. Thanks to its numerous bioactive components, whey is expected to be increasingly incorporated into foods in the future. We investigated the safety of ovine milk whey through in vitro experiments on human primary gingival fibroblast (HGF-1) proliferation and wound healing. Fibroblasts play a crucial role in the repair processes from the late inflammatory phase until the final stages. Cells treated with varying concentrations of ovine whey (0.01%, 0.1%, 1%, and 10%) were able to close wounds more rapidly than vehicle-treated cells. Time- and dose-dependent responses were observed in cell populations exposed to ovine whey. Specifically, wounds treated with 0.1% and 10% milk whey showed better migratory capabilities compared to those treated with 0.01% and 1% milk whey after 24 and 48 h. In addition, ovine milk whey stimulates extracellular matrix deposition, as evidenced by the increasing levels of CD44 antigen density evaluated through FACS analysis, as well as COL1A1 expression measured both via RT-qPCR and immunofluorescence. This phenomenon was particularly evident at concentrations of 0.01% and 10%. Ensuring quality and safety has become a major concern for health authorities in the food industry. Our findings suggest that ovine milk whey is safe and possesses regenerative properties. It facilitates tissue re-establishment following exposure to environmental stress, particularly accelerating gingival wound closure.

Novel insights into the pharmacological modulation of human periodontal ligament stem cells by the amino-bisphosphonate Alendronate.

Alendronate (ALN) is a second-generation bisphosphonate widely used for osteoporosis and cancer-induced bone lesions. Many studies have confirmed a strong relationship between osteonecrosis of the jaws (ONJ) development and oral bisphosphonates, especially ALN, although the molecular mechanisms underlying this pathology have not yet been elucidated. The reduction in bone turnover and vascularization usually observed in ONJ are the result of ALN action on different cell types harboured in oral microenvironment, such as osteoclasts, endothelial cells, and periodontal ligament stem cells (PDLSCs). In this perspective, the present study aims to investigate the effects of different ALN concentrations (2 µM, 5 µM, 10 µM, 25 µM, 50 µM) on the phenotype and functional properties of human PDLSCs (hPDLSCs). hPDLSCs showed a decrease in cell viability (MTT assay) only when treated with ALN concentration of 10 µM or larger for 48 h and 72 h. Cell cycle analysis revealed a moderate increase in proportion of S-phase cells after exposure to low ALN concentration (2-5 µM), an effect that was reverted after exposure to 10-50 µM ALN. Conversely, cell death was evidenced via Annexin V/PI assay at very high concentration of ALN (50 µM) after 4 days of treatment. In addition, we explored whether the effects of ALN on hPDLSCs growth and survival can be mediated by its ability to modulate oxidative stress. To this, we quantified the intracellular ROS amount and lipid peroxidation by using DCF probe and Bodipy staining, respectively. Flow cytometry analysis showed that ALN induced a dose-dependent reduction of intracellular oxidative stress and lipid peroxidation upon treatment with low concentrations at both 48 h and 72 h. Increased levels of oxidative stress was reported at 50 µM ALN and was also confirmed via TEM analysis. Despite the stability of the cellular immunophenotype, hPDLSCs showed impaired mobility after ALN exposure. Chronic exposure (7-14 days) to ALN in the range of 2-10 µM significantly decreased the expression of the differentiation-related factors ALP, RUNX2, COLI, and OPN as well as the osteogenic ability of hPDLSCs compared with untreated cells. Conversely, higher doses were found to be neutral. Our findings indicated that the effects of ALN on hPDLSCs behavior are dose-dependent and suggest a role for oxidative stress in ALN-induced cell death that may lead to novel therapeutic approaches for ONJ.

Utilization of Dairy By-Products as a Source of Functional and Health Compounds-The Role of Ovine Colostrum and Milk Whey on Chronic Myeloid Leukemia Cells.

Nowadays, the search for food products that promote consumers' health has gained interest, and dairy by-products, due to their biological quality, could have a prominent position among products with health benefits. However, little is known about their activity on cancer cells. This study aimed to provide evidence about the effect of ovine colostrum and milk whey on K562 cells, a model of the human chronic myeloid leukemia cell line. The exposure of K562 cells to a single administration of sheep by-products at different concentrations for three days and three treatments for three days was carried out. Using a flow cytometric approach, we found that CD235a expression remained stable in the cells exposed to ovine whey (milk and colostrum) at concentrations ranging from 1 ng/mL to 100 µg/mL, after three days from one or three administrations, respectively. A significant reduction in fluorescent cells was observed in the populations exposed to 1 mg/mL of both milk and colostrum at the same time points. In these conditions, the size and granularity of the leukemic cells also changed, with a substantial reduction in the number of actively dividing cells in the S phase of the cell cycle. This phenomenon was highlighted by the Annexin V/PI cytofluorimetric test, which is able to provide quantitative results regarding the population of cells in early or late apoptosis or necrotic cells after exposure to a single dose or three doses of colostrum or sheep whey for three days, respectively. This report showed that both colostrum and milk whey were able to modify the phenotypic profile and cell cycle of the K562 cell line, inducing apoptosis at the highest concentration.

A Review of Novel Strategies for Human Periodontal Ligament Stem Cell Ex Vivo Expansion: Are They an Evidence-Based Promise for Regenerative Periodontal Therapy?

Periodontitis is a gingiva disease sustained by microbially associated and host-mediated inflammation that results in the loss of the connective periodontal tissues, including periodontal ligament and alveolar bone. Symptoms include swollen gingiva, tooth loss and, ultimately, ineffective mastication. Clinicians utilize regenerative techniques to rebuild and recover damaged periodontal tissues, especially in advanced periodontitis. Human periodontal ligament stem cells (hPDLSCs) are considered an appealing source of stem cells for regenerative therapy in periodontium. hPDLSCs manifest the main properties of mesenchymal stem cells, including the ability to self-renew and to differentiate in mesodermal cells. Significant progress has been made for clinical application of hPDLSCs; nevertheless, some problems remain, including the small number of cells isolated from each sample. In recent decades, hPDLSC ex vivo expansion and differentiation have been improved by modifying cell culture conditions, especially with the supplementation of cytokines' or growth factors' mix, chemicals, and natural compounds, or by using the decellularized extracellular matrix. Here, we analyzed the changes in stemness properties and differentiation potential of hPDLSCs when culturing in alternative media. In addition, we focused on the possibility of replacing FBS with human emoderivates to minimize the risks of xenoimmunization or zoonotic transmission when cells are expanded for therapeutic purposes.

Emotional and Spontaneous Locomotor Behaviors Related to cerebellar Daidzein-dependent TrkB Expression Changes in Obese Hamsters.

Current evidence supports the beneficial role of phytoestrogens in metabolic diseases, but their influences on spontaneous motor and anxiety behaviors plus neuroprotective effects have still not been completely elucidated. With the present study, neuro-behavioral activities were correlated to daidzein (DZ)-dependent expression changes of a high affinity catalytic receptor for several neurotrophins, and namely tropomyosin-related kinase B receptor (TrkB) in the cerebellar cortex of high-fat diet (HFD) hamsters (Mesocricetus auratus). Indeed, these changes appear to be tightly linked to altered plasma lipid profiles as shown by reduced low-density lipoproteins plus total cholesterol levels in DZ-treated obesity hamsters accounting for increased spontaneous locomotor together with diminished anxiety activities in novel cage (NCT) and light/dark box (LDT) tests. For this latter case, the anxiolytic-like hamsters spent more time in the light compartment, which was retained the aversive area of the LDT box. As for the evaluation of the neurotrophin receptor site, significantly elevated TrkB levels were also detected, for the first time, in the cerebellum of obese hamsters treated with DZ. In this condition, such a treatment widely led to an overall improvement of HFD-induced neurodegeneration damages, above all in the Purkinje and granular layers of the cerebellum. In this context, the notably active TrkB signaling events occurring in a DZ-dependent manner may turn out to be a key neuroprotective element capable of restoring normal emotional and spontaneously linked locomotor behaviors regulated by cerebellar cortical areas especially in obesity-related conditions.

The anticancer effects of Metformin in the male germ tumor SEM-1 cell line are mediated by HMGA1.

Introduction: Germ cell tumors (GCTs) are the most common type of cancer in young men. These tumors usually originate from the testis, but they can occasionally develop from extragonadal sites probably due to primordial germ cells (PGCs) migration errors. Cisplatin-based chemotherapy is usually effective for male GCTs, but the risk of toxicity is high and new therapeutic strategies are needed. Although Metformin (Met) has been widely studied as a potential cancer treatment over the past decades, there is limited evidence to support its use in treating male GCTs. Additionally, the mechanism by which it acts on tumor cells is still not entirely understood. Methods: SEM-1 cells, a newly established human cell line of extragonadal origin, were treated with Met. Cell viability was studied by MTT assay, while cell migration and invasion were studied by the wound healing assay and the transwell assay, respectively. The effect of Met on 3D spheroid formation was determined by seeding SEM-1 cells in appropriate cell suspension culture conditions, and cell cycle was characterized by flow cytometry. Factors involved in PGCs migration and GCT invasion, such as IGFBP1, IGF1R, MMP-11 and c-Kit, together with cyclin D1 (a key regulator of cell cycle progression), and the upstream factor, HMGA1, were determined by immunoblots. Results: Treatment of SEM-1 cells with Met resulted in a potent and dose-dependent reduction of cell proliferation, as evidenced by decreased nuclear abundance of cyclin D1 and cell cycle arrest in G1 phase. Also, Met prevented the formation of 3D spheroids, and blocked cell migration and invasion by reducing the expression of IGFBP1, IGF1R and MMP-11. Both, IGFBP1 and MMP-11 are under control of HMGA1, a chromatin-associated protein that is involved in the regulation of important oncogenic, metabolic and embryological processes. Intriguingly, an early reduction in the nuclear abundance of HMGA1 occurred in SEM-1 cells treated with Met. Conclusions: Our results document the antiproliferative and antimigratory effects of Met in SEM-1 cells, providing new insights into the potential treatments for male GCTs. The anticancer properties of Met in SEM-1 cells are likely related to its ability to interfere with HMGA1 and downstream targets, including cyclin D1, the IGFs system, and MMP-11.

Targeting of Mevalonate-Isoprenoid Pathway in Acute Myeloid Leukemia Cells by Bisphosphonate Drugs.

Metabolic reprogramming represents a hallmark of tumorigenesis to sustain survival in harsh conditions, rapid growth and metastasis in order to resist to cancer therapies. These metabolic alterations involve glucose metabolism, known as the Warburg effect, increased glutaminolysis and enhanced amino acid and lipid metabolism, especially the cholesterol biosynthesis pathway known as the mevalonate pathway and these are upregulated in several cancer types, including acute myeloid leukemia (AML). In particular, it was demonstrated that the mevalonate pathway has a pivotal role in cellular transformation. Therefore, targeting this biochemical process with drugs such as statins represents a promising therapeutic strategy to be combined with other anticancer treatments. In the last decade, several studies have revealed that amino-bisphosphonates (BP), primarily used for bone fragility disorders, also exhibit potential anti-cancer activity in leukemic cells, as well as in patients with symptomatic multiple myeloma. Indeed, these compounds inhibit the farnesyl pyrophosphate synthase, a key enzyme in the mevalonate pathway, reducing isoprenoid formation of farnesyl pyrophosphate and geranylgeranyl pyrophosphate. This, in turn, inhibits the prenylation of small Guanosine Triphosphate-binding proteins, such as Ras, Rho, Rac, Rab, which are essential for regulating cell survival membrane ruffling and trafficking, interfering with cancer key signaling events involved in clonal expansion and maturation block of progenitor cells in myeloid hematological malignancies. Thus, in this review, we discuss the recent advancements about bisphosphonates' effects, especially zoledronate, analyzing the biochemical mechanisms and anti-tumor effects on AML model systems. Future studies will be oriented to investigate the clinical relevance and significance of BP treatment in AML, representing an attractive therapeutic strategy that could be integrated into chemotherapy.

Correlation of distinct behaviors to the modified expression of cerebral Shank1,3 and BDNF in two autistic animal models.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder featuring altered neuronal circuitry and consequently impaired social interactions, restrictive interests plus repetitive stereotypic activities. In the present study, differentiated behaviors of valproic (VPA) and propionic (PPA) acid-mediated autism rats were correlated to cerebral scaffolding proteins (Shank1,3) and BDNF expression variations. Sprague-Dawley offspring that received VPA during pregnancy displayed a notably diminished permanence (-78 %, p < 0.01) in the light chamber of light dark (LD) test, reduced exploratory tasks, i.e. grooming (-90 %) and rearing (-65 %). Moreover, they executed extremely greater climbing intervals (+300 %, p < 0.001) in novel cage (NC) test, plus exhibited an extremely reduced (-331 %) discrimination index in novel object recognition (NOR) test when compared to controls. PPA-treated postnatal days (PND) 12-16 rats also displayed anxiety-like behaviors, although in a less evident manner, as indicated by a moderate time (+55 %; p < 0.05) spent in dark chamber along with notable and moderate decreases in digging (-78 %) plus grooming (-52 %), respectively. Contextually, VPA- more than PPA supplied opposite Shank1,3 expression changes in cerebellum (CB; -62 %; +78 %), dorsomedial prefrontal cortex (DM-PFC; +95 % -76 %), respectively, while resulting extremely upregulated in hippocampus (HIP; +125 % - +155 %). Even BDNF resulted to be substantially and notably diminished in HIP (-85 %) and DM-PFC (-72 %), respectively, of VPA rats while it was only moderately reduced (-35 % to -45 %) in these same areas of PPA rats. The early altered brain-specific expression levels accounting for different behavioral performances may provide useful diagnostic indications and constitute valuable therapeutic strategies for autistic patients.

Extracellular Matrix in Calcific Aortic Valve Disease: Architecture, Dynamic and Perspectives.

Calcific Aortic Valve Disease (CAVD) is the most common valvular heart disease in developed countries and in the ageing population. It is strongly correlated to median age, affecting up to 13% of the population over the age of 65. Pathophysiological analysis indicates CAVD as a result of an active and degenerative disease, starting with sclerosis and chronic inflammation and then leaflet calcification, which ultimately can account for aortic stenosis. Although CAVD has been firstly recognized as a passive event mostly resulting from a degenerative aging process, much evidences suggests that calcification arises from different active processes, involving both aortic valve-resident cells (valve endothelial cells, valve interstitial cells, mesenchymal stem cells, innate immunity cells) and circulating cells (circulating mesenchymal cells, immunity cells). Moreover, a role for the cell-derived "matrix vesicles" and extracellular matrix (ECM) components has also been recognized. The aim of this work is to review the cellular and molecular alterations occurring in aortic valve during CAVD pathogenesis, focusing on the role of ECM in the natural course of the disease.

Daidzein Pro-cognitive Effects Coincided with Changes of Brain Neurotensin1 Receptor and Interleukin-10 Expression Levels in Obese Hamsters.

At present, concerns are pointing to "tasteful" high-fat diets as a cause of conditioning physical-social states that through alterations of some key emotional- and nutritional-related limbic circuits such as hypothalamic and amygdalar areas lead to obesity states. Feeding and energetic homeostatic molecular mechanisms are part of a complex neuronal circuit accounting for this metabolic disorder. In an attempt to exclude conventional drugs for treating obesity, daidzein, a natural glycosidic isoflavone, which mimics estrogenic neuroprotective properties against increased body weight, is beginning to be preferred. In this study, evident anxiolytic-like behaviors were detected following treatment of high-fat diet hamsters with daidzein as shown by extremely evident (p < 0.001) exploration tendencies in novel object recognition test and a notably greater amount of time spent (p < 0.01) in open arms of elevated plus maze. Moreover, the isoflavone promoted a protective role against neurodegeneration processes as shown by few, if any, amino cupric silver granules in amygdalar, hypothalamic and hippocampal neuronal fields when compared with obese hamsters. Interestingly, elevated expression levels of the anorexic neuropeptide receptor neurotensin1 in the above limbic areas of obese hamsters were extremely reduced by daidzein, especially during recovery of cognitive events. Contextually, such effects were strongly paralleled by increased levels of the anti-neuroinflammatory cytokine, interleukin-10. Our results corroborate a neuroprotective ability of this natural glycosidic isoflavone, which through its interaction with the receptor neurotensin1 and interleukin-10 pathways is correlated not only to improved feeding states, and subsequently obesity conditions, but above all to cognitive performances.

Impact of Magnetic Stimulation on Periodontal Ligament Stem Cells.

The aim of this study was to evaluate the effect of a time-dependent magnetic field on the biological performance of periodontal ligament stem cells (PDLSCs). A Western blot analysis and Alamar Blue assay were performed to investigate the proliferative capacity of magnetically stimulated PDLSCs (PDLSCs MAG) through the study of the MAPK cascade (p-ERK1/2). The observation of ALP levels allowed the evaluation of the effect of the magnetic field on osteogenic differentiation. Metabolomics data, such as oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and ATP production provided an overview of the PDLSCs MAG metabolic state. Moreover, the mitochondrial state was investigated through confocal laser scanning microscopy. Results showed a good viability for PDLSCs MAG. Magnetic stimulation can activate the ERK phosphorylation more than the FGF factor alone by promoting a better cell proliferation. Osteogenic differentiation was more effectively induced by magnetic stimulation. The metabolic panel indicated significant changes in the mitochondrial cellular respiration of PDLSCs MAG. The results suggested that periodontal ligament stem cells (PDLSCs) can respond to biophysical stimuli such as a time-dependent magnetic field, which is able to induce changes in cell proliferation and differentiation. Moreover, the magnetic stimulation also produced an effect on the cell metabolic profile. Therefore, the current study demonstrated that a time-dependent magnetic stimulation may improve the regenerative properties of PDLSCs.

The Case of Medication-Related Osteonecrosis of the Jaw Addressed from a Pathogenic Point of View. Innovative Therapeutic Strategies: Focus on the Most Recent Discoveries on Oral Mesenchymal Stem Cell-Derived Exosomes.

Bisphosphonates-related osteonecrosis of the jaw (BRONJ) was firstly reported by Marx in 2003. Since 2014, the term medication-related osteonecrosis of the jaw (MRONJ) is recommended by the American Association of Oral and Maxillofacial Surgeons (AAOMS). Development of MRONJ has been associated to the assumption of bisphosphonates but many MRONJ-promoting factors have been identified. A strong involvement of immunity components has been suggested. Therapeutic intervention includes surgical and non-surgical treatments, as well as regenerative medicine procedures for the replacement of the lost tissues. The literature confirms that the combination of mesenchymal stem cells (MSCs), biomaterials and local biomolecules can support the regeneration/repair of different structures. In this review, we report the major open topics in the pathogenesis of MRONJ. Then, we introduce the oral tissues recognized as sources of MSCs, summing up in functional terms what is known about the exosomes release in physiological and pathological conditions.

Dose-Dependent Effects of Zoledronic Acid on Human Periodontal Ligament Stem Cells: An In Vitro Pilot Study.

Bisphosphonates (BPs) are widely used to treat several metabolic and oncological diseases affecting the skeletal system. Despite BPs' well-known therapeutic potential, they also displayed important side effects, among which is BPs-related osteonecrosis of the jaw, by targeting osteoclast activities, osteoblast, and osteocyte behavior. The aim of this study is to evaluate the biological effects of zoledronic acid (ZOL) in an in vitro model of periodontal ligament stem cells (PDLSCs) by using an experimental setting that resembles the in vivo conditions. PDLSCs were treated with different concentrations of ZOL ranging from 0.1 to 5 µM. The effects of ZOL exposure were evaluated on cell viability via 3-[4,5-Dimethylthiaoly]-2,5-diphenyltetrazolium bromide (MTT), cell cycle analysis, apoptosis detection, and immunofluorescence. Quantitative real-time polymerase chain reaction (PCR), colorimetric detection of alkaline phosphatase activity, and Alizarin Red S staining were performed to investigate the osteogenic potential of PDLSCs exposed to ZOL. MTT analysis showed that the viability of PDLSCs exposed to ZOL concentration ≥1.5 µM for 3 and 6 days was significantly lower (P < 0.001) than that of untreated cells. The percentage of apoptotic cells was significantly higher in PDLSCs exposed for 4 days to ZOL at 2 µM (P < 0.01) and 5 µM (P < 0.001) when compared to the control. Moreover, ZOL treatment (3 days) accounted for alterations in cell cycle distribution, with an increase in the proportion of cells in G0/G1 phase and a reduction in the proportion of cells in S phase. Chronic exposure (longer than 7 days) of PDLSCs to ZOL accounted for the downregulation of ALP, RUNX2, and COL1 genes at all tested concentrations, which fit well with the reduced alkaline phosphatase activity reported after 7 and 14 days of treatment. Reduced Col1 deposition in the extracellular matrix was reported after 14 days of treatment. Increased calcium deposits were observed in treated cells when compared to the control cultures. In conclusion, chronic exposure to 1 µM ZOL induced significant reduction of osteogenic differentiation, while ZOL concentrations ≥1.5 µM are required to impair PDLSCs viability and induce apoptosis.

FtH-Mediated ROS Dysregulation Promotes CXCL12/CXCR4 Axis Activation and EMT-Like Trans-Differentiation in Erythroleukemia K562 Cells.

The cell-microenvironment communication is essential for homing of hematopoietic stem cells in stromal niches. Recent evidences support the involvement of epithelial-to-mesenchymal (EMT) process in hematopoietic stem cell homeostasis as well as in leukemia cells invasiveness and migration capability. Here, we demonstrate that the alteration of iron homeostasis and the consequent increase of redox metabolism, mediated by the stable knock down of ferritin heavy chain (FtH), enhances the expression of CXCR4 in K562 erythroleukemia cells, thus promoting CXCL12-mediated motility. Indeed, addition of the CXCR4 receptor antagonist AMD3100 reverts this effect. Upon FtH knock down K562 cells also acquire an "EMT-like" phenotype, characterized by the increase of Snail, Slug and Vimentin with the parallel loss of E-cadherin. By using fibronectin as substrate, the cell adhesion assay further shows a reduction of cell adhesion capability in FtH-silenced K562 cells. Accordingly, confocal microscopy shows that adherent K562 control cells display a variety of protrusions while FtH-silenced K562 cells remain roundish. These phenomena are largely due to the reactive oxygen species (ROS)-mediated up-regulation of HIF-1α/CXCR4 axis which, in turn, promotes the activation of NF-κB and the enhancement of EMT features. These data are confirmed by treatments with either N-acetylcysteine (NAC) or AMD3100 or NF-κB inhibitor IκB-alpha which revert the FtH-silenced K562 invasive phenotype. Overall, our findings demonstrate the existence of a direct relationship among iron metabolism, redox homeostasis and EMT in the hematological malignancies. The effects of FtH dysregulation on CXCR4/CXCL12-mediated K562 cell motility extend the meaning of iron homeostasis in the leukemia cell microenvironment.

H-Ferritin Affects Cisplatin-Induced Cytotoxicity in Ovarian Cancer Cells through the Modulation of ROS.

Reactive oxygen species (ROS) mediates cisplatin-induced cytotoxicity in tumor cells. However, when cisplatin-induced ROS do not reach cytotoxic levels, cancer cells may develop chemoresistance. This phenomenon can be attributed to the inherited high expression of antioxidant protein network. H-Ferritin is an important member of the antioxidant system due to its ability to store iron in a nontoxic form. Altered expression of H-Ferritin has been described in ovarian cancers; however, its functional role in cisplatin-based chemoresistance of this cancer type has never been explored. Here, we investigated whether the modulation of H-Ferritin might affect cisplatin-induced cytotoxicity in ovarian cancer cells. First, we characterized OVCAR3 and OVCAR8 cells for their relative ROS and H-Ferritin baseline amounts. OVCAR3 exhibited lower ROS levels compared to OVCAR8 and greater expression of H-Ferritin. In addition, OVCAR3 showed pronounced growth potential and survival accompanied by the strong activation of pERK/pAKT and overexpression of c-Myc and cyclin E1. When exposed to different concentrations of cisplatin, OVCAR3 were less sensitive than OVCAR8. At the lowest concentration of cisplatin (6 µM), OVCAR8 underwent a consistent apoptosis along with a downregulation of H-Ferritin and a consistent increase of ROS levels; on the other hand, OVCAR3 cells were totally unresponsive, H-Ferritin was almost unaffected, and ROS amounts met a slight increase. Thus, we assessed whether the modulation of H-Ferritin levels was able to affect the cisplatin-mediated cytotoxicity in both the cell lines. H-Ferritin knockdown strengthened cisplatin-mediated ROS increase and significantly restored sensitivity to 6 µM cisplatin in resistant OVCAR3 cells. Conversely, forced overexpression of H-Ferritin significantly suppressed the cisplatin-mediated elevation of intracellular ROS subsequently leading to a reduced responsiveness in OVCAR8 cells. Overall, our findings suggest that H-Ferritin might be a key protein in cisplatin-based chemoresistance and that its inhibition may represent a potential approach for enhancing cisplatin sensitivity of resistant ovarian cancer cells.

MicroRNA let-7g acts as tumor suppressor and predictive biomarker for chemoresistance in human epithelial ovarian cancer.

Remarkable deregulation of microRNAs has been demonstrated in epithelial ovarian cancer (EOC). In particular, some of the let-7 miRNA family members have been proposed as tumor suppressors. Here, we explored the functional roles of let-7g in EOC. The ectopic overexpression of let-7g in OVCAR3 and HEY-A8 EOC cells induced i) a down-regulation of c-Myc and cyclin-D2 thus promoting cell cycle arrest, ii) a reduction of Vimentin, Snail and Slug thus counteracting the progression of epithelial to mesenchymal transition, iii) a chemosensitization to cis-platinum treatment. Next, analysis of human EOC tissues revealed that let-7g expression was significantly reduced in tumor tissue specimens of patients with EOC compared to their non-tumor counterparts (p = 0.0002). Notably, low let-7g tissue levels were significantly associated with acquired chemoresistance of patients with late-stage of EOC (n = 17, p = 0.03194). This finding was further validated in the serum samples collected from the same cohort of patients (n = 17, p = 0.003). To conclude, we demonstrate that let-7g acts as tumor suppressor and might be used to disable EOC tumor progression and chemoresistance to cis-platinum-based chemotherapy. Furthermore, we propose that decreased expression of let-7g could serve as a tissue and serum biomarker able to predict the chemo-resistant features of EOC patients.

Identification of mesothelial cells in intraoperative blood salvage.

Intraoperative auto-transfusion with the use of cell saver systems is routinely used to reduce the rate of packed red blood transfusion in major surgery. Nevertheless some concerns have been raised on possible risks of coagulation disorders. The aim of the study was to analyze the blood processed by the cell saver, ready to be re-infused to the patient, in order to individuate unexpected cellular components, that can favor coagulopathy. We tested the blood processed by the cell saver in thirteen patients undergoing coronary bypass surgery with Cellsearch®, ScreenCell®, Cytology and Immunofluorescence. Those four methods allowed us to look for the presence of unexpected cells, quantify and characterize them. Furthermore, the blood processed by the cell saver was mixed with the patient's peripheral blood and analyzed with the ROTEM® thromboelastography. The Cellsearch® revealed and counted a mean number of 1241 unexpected cells/7.5 ml in the blood processed by the cell saver. The ScreenCell® and Cytology confirmed the presence of non-hematological cells. Immunofluorescence showed positivity for Calretinin and WT-1, confirming the mesothelial origin. Moreover we detected a peculiar arrangement of the platelets around the mesothelial cells in a "cloud" form, suggesting platelet activation. The ROTEM® analysis showed a significantly longer clot formation time, smaller clot amplitude and maximum clot firmness, compared to controls. In conclusion we demonstrated the presence of mesothelial cells in the cell saving blood, ready to be auto-transfused. This finding can contribute to develop a platelet depletion coagulopathy, with coagulation factors consumption.

Trabectedin triggers direct and NK-mediated cytotoxicity in multiple myeloma.

BACKGROUND: Genomic instability is a feature of multiple myeloma (MM), and impairment in DNA damaging response (DDR) has an established role in disease pathobiology. Indeed, a deregulation of DNA repair pathways may contribute to genomic instability, to the establishment of drug resistance to genotoxic agents, and to the escape from immune surveillance. On these bases, we evaluated the role of different DDR pathways in MM and investigated, for the first time, the direct and immune-mediated anti-MM activity of the nucleotide excision repair (NER)-dependent agent trabectedin. METHODS: Gene-expression profiling (GEP) was carried out with HTA2.0 Affymetrix array. Evaluation of apoptosis, cell cycle, and changes in cytokine production and release have been performed in 2D and 3D Matrigel-spheroid models through flow cytometry on MM cell lines and patients-derived primary MM cells exposed to increasing nanomolar concentrations of trabectedin. DNA-damage response has been evaluated through Western blot, immunofluorescence, and DNA fragmentation assay. Trabectedin-induced activation of NK has been assessed by CD107a degranulation. miRNAs quantification has been done through RT-PCR. RESULTS: By comparing GEP meta-analysis of normal and MM plasma cells (PCs), we observed an enrichment in DNA NER genes in poor prognosis MM. Trabectedin triggered apoptosis in primary MM cells and MM cell lines in both 2D and 3D in vitro assays. Moreover, trabectedin induced DDR activation, cellular stress with ROS production, and cell cycle arrest. Additionally, a significant reduction of MCP1 cytokine and VEGF-A in U266-monocytes co-cultures was observed, confirming the impairment of MM-promoting milieu. Drug-induced cell stress in MM cells led to upregulation of NK activating receptors ligands (i.e., NKG2D), which translated into increased NK activation and degranulation. Mechanistically, this effect was linked to trabectedin-induced inhibition of NKG2D-ligands negative regulators IRF4 and IKZF1, as well as to miR-17 family downregulation in MM cells. CONCLUSIONS: Taken together, our findings indicate a pleiotropic activity of NER-targeting agent trabectedin, which appears a promising candidate for novel anti-MM therapeutic strategies.

In Vitro Long-Term Expansion and High Osteogenic Potential of Periodontal Ligament Stem Cells: More Than a Mirage.

The periodontal ligament displays a reservoir of mesenchymal stem cells which can account for periodontal regeneration. Despite the numerous studies directed at the definition of optimal culture conditions for long-term expansion of periodontal ligament stem cells (PDLSCs), no consensus has been reached as to what is the ideal protocol. The aim of the present study was to determine the optimal medium formulation for long-term expansion and stemness maintenance of PDLSCs, in order to obtain a sufficient number of cells for therapeutic approaches. For this purpose, the effects of three different culture medium formulations were evaluated on PDLSCs obtained from three periodontal ligament samples of the same patient: minimum essential medium Eagle, alpha modification (α-MEM), Dulbecco's modified Eagle's medium (DMEM), both supplemented with 10% fetal bovine serum (FBS), and a new medium formulation, Ham's F12 medium, supplemented with 10% FBS, heparin 0.5 U/ml, epidermal growth factor (EGF) 50 ng/ml, fibroblast growth factor (FGF) 25 ng/ml, and bovine serum albumin (BSA) 1% (enriched Ham's F12 medium; EHFM). PDLSCs grown in EHFM displayed a higher PE-CD73 mean fluorescence intensity compared with cells maintained in α-MEM and DMEM, even at later passages. Cells maintained in EHFM displayed an increased population doubling and a reduced population doubling time compared with cells grown in DMEM or α-MEM. α-MEM, DMEM and EHFM with added dexamethasone, 2-phospho-L-ascorbic acid, and ß-glycerophosphate were all able to promote alkaline phosphatase activity; however, no calcium deposition was detected in PDLSCs cultured in EHFM-differentiation medium. When EHFM-, α-MEM- and DMEM-expanded PDLSCs were transferred to a commercial culture medium for the osteogenesis, mineralization became much more evident in confluent monolayers of EHFM-expanded PDLSCs compared with DMEM and α-MEM. The results suggest EHFM is the optimal medium formulation for growth and stemness maintenance of primary PDLSCs. Moreover, EHFM confers higher osteogenic potential to PDLSCs compared with cells maintained in the other culture media. Overall, the results of the present work confirmed the advantages of using EHFM for long-term expansion of mesenchymal cells in vitro and the preservation of high osteogenic potential.