Characterization and gene expression of cancer stem cells grown as sarcospheres from human primary sarcomas

This study aimed to identify, isolate and characterize cancer stem cells from human primary sarcomas. We performed cytometric analyses for stemness and differentiation antigens including CD29, CD34, CD44, CD90, CD117 and CD133 on 21 human primary sarcomas on the day of surgery. From sarcoma biopsies, we obtained two chondrosarcoma stabilized cell lines and two osteosarcoma stabilized cell lines on which spheres formation, side population profile, stemness gene expression and in vivo and in vitro assays were performed. On a chondrosarcoma cell line, the whole genoma microarray analyses were performed (sarcospheres versus adherent cells). All samples expressed the CD133, CD44 and CD29 markers. We selected a CD133+ subpopulation from stabilized cell lines that displayed the capacity to grow as sarcospheres able to initiate and sustain tumour growth in NOD/SCID mice, to express stemness genes including OCT3/4, Nanog, Sox2 and Nestin and to differentiate into mesenchymal lineages. Microarray analyses pointed out a huge gene expression difference between sarcospheres and adherent cells. About 2000 genes, including ones related to cell cycle, stemness and epigenetic regulation, resulted to be very differentially expressed in the two population considered. With signed ratio of 2,806 genes were over-expressed and 1,029 under-expressed on sarcospheres when compared with adherent cells. The most highly overexpressed gene were thioredoxin interacting protein (fold +25) that modulates the cellular redox state, growth differentiation factor 15 (fold +10), member of the TGF–β superfamily , histone cluster 1, H2ad (fold +9) and solute carrier family 2 member 14 that facilitate glucose transport (fold +8,9). Our findings evidence the existence of cancer stem cells in human primary bone sarcomas and highlight CD133 as pivotal marker for identification of these cells. This may be of primary importance in the development of new therapeutic strategies and new prognostic procedures against these highly aggressive and metastatic tumours

Polyblend Nanofibers to Regenerate Gingival Tissue: A Preliminary In Vitro Study

Aim: The regeneration of small periodontal defects has been considered an important divide and challenging issue for dental practitioners. The aim of this preliminary in vitro study was to analyze the effects of polycaprolactone (PCL) nanofibers enriched with hyaluronic acid and vitamin E vs. nude nanofibers on gingival fibroblasts activity, an innovative graft for periodontal soft tissue regeneration purposes. Methods: Nanofibers were produced in PCL (NF) or PCL enriched with hyaluronic acid and vitamin E (NFE) by electrospinning technique. NF and NFE were stereologically and morphologically characterized by scanning electron microscope (SEM), and composition was analyzed by infrared spectroscopy. Human fibroblasts were obtained from one gingival tissue fragment (HGF) and then seeded on NF, NFE, and plastic (CT). Cell adhesion and morphology were evaluated using SEM at 24 h and cell viability after 24, 48, and 72 h by alamarBlue® assay. Gene expression for COL-I, LH2b, TIMP-1, PAX, and VNC was analyzed by real-time RT-PCR in samples run in triplicate and GAPDH was used as housekeeping gene. Slot blot analysis was performed and immunoreactive bands were revealed for MMP-1 and COL-I. YAP and p-YAP were analyzed by Western blot and membranes were reprobed by α-tubulin. Statistical analysis was performed. Results: IR spectrum revealed the presence of PCL in NF and PCL and vitamin E and hyaluronic acid in NFE. At 24 h, HGF adhered on NF and NFE conserving fibroblast like morphology. At 72 h from seeding, statistically significant differences were found in proliferation of HGF cultured on NF compared to NFE. Expression of genes (LH2b, TIMP-1, and MMP-1) and proteins (COL-I) related to collagen turnover revealed a reduction of COL-1 secretion in cells cultured on NF and NFE compared to CT; however, NFE stimulated cross-linked collagen deposition. Mechanosensor genes (PAX, VNC, and YAP) were upregulated in HGF on NF while they were decreased in cells grown on NFE. Conclusion: Preliminary data suggest that PCL-enriched nanofibers could represent a support to induce HGF proliferation, adhesion, collagen cross-linking, and to reduce collagen degradation, therefore favoring collagen deposition in gingival connective tissue

Methylation and epigenetic modification by 5’ azacytidine and valproic acid treatment increase stemness attributes in bone sarcoma cell lines

Bone sarcoma is an aggressive malignancy with high mortality rate. Despite recent advances, the prognosis is still extremely poor. Bone sarcomas contain a small cell population with stem cell like properties, referred to as cancer stem cells (CSCs) expressing CD133 (Tirino et al, 2009; 2011). The biological relevance and regulatory mechanism of CD133 expression are not yet understood. The aim of this study is to elucidate mechanisms regulating aberrant expression of CD133 and stemness phenotype. Saos-2, MG63 and BS15 cell lines were treated with 0,5 mM valproic acid (VPA) and 3μM 5’azacytidine (5-AZA) for 48 hours alone and in combination. CD133 and stemness markers expression including OCT4, Sox2 and Nanog were analyzed by flow cytometry and real-time PCR. Vimentin and osteocalcin levels were also tested. Sarcospheres formation rate was assessed as spheres number/seed single cell number. After treatment with 5-AZA or VPA, the expression level of CD133 mRNA as well as of protein was significantly increased in all three cell lines. Also OCT4, Sox2 and Nanog, stemness markers, and vimentin, mesenchymal marker resulted to be upregulated after treatment by real time-PCR. On the contrary, the expression level of osteocalcin remained similar before and after treatment. Interestingly, combined treatment with 5-AZA and VPA induced an increase of CD133 expression in a synergistic manner in all three cell lines. In addition, sarcospheres formation rate was increased after drug treatment compared to untreated cells. Also in this case, the drug combination lead to synergistic increase of formation rate of spheres. In conclusion, our results indicate that DNA methylation is an important determinant of CD133 and stemness profile in human bone sarcomas and this mechanism may be associated with histone deacetilase inhibition

Cancer stem cells in head and neck tumors: evidence for metastatic spread and treatment resistance

The major challenge in the management of patients with oral squamous cell carcinomas (OSCC) is the development of resistance to therapy leading to disseminated disease. Since cancer stem cells (CSC) have emerged as important players in OSCC metastasis, our objectives were to explore the implications of CSC in OSCC tumor progression, invasion and response to conventional therapies. Methods: A panel of well-characterized cell lines originated from the most common sites in the head and neck area was used. Cells were cultured as floating spheres or under normal adherent conditions and analyzed for CD44, ALDH, CD24, CD29, CD56 by flow cytometry, PCR arrays for genes related to stemness, metastasis and EMT . We also investigated sLeX expression, known to play a key-role in many cancers metastasis by promoting tumor cells binding to endothelial E-selectin. We analyzed the tumorigenic potential of OSCC cells by invasion assays and in vivo OSCC experimental models comparatively to CSC cells. Moreover resistance to cisplatin and radiation was assessed by annexin V/PI assay and by colony forming assay. Results: The highest levels of sLeX expression were found in cell lines originated from oral cavity (9%-47%) compared to other head and neck locations (0.1%-7%). Cells grown as spheres were 95-100% positive for sLeX compared to 10-40% of adherent counterpart. Although sLeX+ and sLeX- cells were both able to form spheres, sLeX+ spheres were predominant and larger. Flow cytometry and PCR arrays indicated that the spheres were highly enriched in CSC and metastatic markers. Consistently, the spheres showed increased invasive and tumorigenic potential, and resistance to conventional chemotherapy and radiations. Conclusion: these studies are the first to unveil a novel link between sLeX expression, stem cell formation and metastatic spread in OSCC, and provide supportive evidence for CSC resistance to treatment. Understanding the mechanisms of tumor invasion and metastasis will improve patient outcome and survival

Altered gut microbiota and endocannabinoid system tone in vitamin D deficiency-mediated chronic pain

Abstract Recent evidence points to the gut microbiota as a regulator of brain and behavior, although it remains to be determined if gut bacteria play a role in chronic pain. The endocannabinoid system is implicated in inflammation and chronic pain processing at both the gut and central nervous system (CNS) levels. In the present study, we used low Vitamin D dietary intake in mice and evaluated possible changes in gut microbiota, pain processing and endocannabinoid system signaling. Vitamin D deficiency induced a lower microbial diversity characterized by an increase in Firmicutes and a decrease in Verrucomicrobia and Bacteroidetes. Concurrently, vitamin D deficient mice showed tactile allodynia associated with neuronal hyperexcitability and alterations of endocannabinoid system members (endogenous mediators and their receptors) at the spinal cord level. Changes in endocannabinoid (anandamide and 2-arachidonoylglycerol) levels were also observed in the duodenum and colon. Remarkably, the anti-inflammatory anandamide congener, palmitoylethanolamide, counteracted both the pain behaviour and spinal biochemical changes in vitamin D deficient mice, whilst increasing the levels of Akkermansia, Eubacterium and Enterobacteriaceae, as compared with vehicle-treated mice. Finally, induction of spared nerve injury in normal or vitamin D deficient mice was not accompanied by changes in gut microbiota composition. Our data suggest the existence of a link between Vitamin D deficiency – with related changes in gut bacterial composition – and altered nociception, possibly via molecular mechanisms involving the endocannabinoid and related mediator signaling systems

The trans-subclavian retrograde approach for transcatheter aortic valve replacement: Single-center experience

ObjectiveAortic valve disease is the most common acquired valvular heart disease in adults. With the increasing elderly population, the proportion of patients with symptomatic aortic stenosis who are unsuitable for conventional surgery is increasing. Transcatheter aortic valve implantation has rapidly gained credibility as a valuable alternative to surgery to treat these patients; however, they often have severe iliac-femoral arteriopathy, which renders the transfemoral approach unusable. We report our experience with the trans-subclavian approach for transcatheter aortic valve implantation using the CoreValve (Medtronic CV Luxembourg S.a.r.l.) in 6 patients.MethodsIn May 2008 to September 2009, 6 patients (mean age of 82 ± 5 years), with symptomatic aortic stenosis and no reasonable surgical option because of excessive risk, were excluded from percutaneous femoral CoreValve implantation because of iliac-femoral arteriopathy. These patients underwent transcatheter aortic valve implantation via the axillary artery. Procedures were performed by a combined team of cardiologists, cardiac surgeons, and anesthetists in the catheterization laboratory. The CoreValve 18F delivery system was introduced via the left subclavian artery in 6 patients, 1 with a patent left internal thoracic to left anterior descending artery graft.ResultsProcedural success was obtained in all patients, and the mean aortic gradient decreased 5 mm Hg or less immediately after valve deployment. One patient required implantation of a permanent pacemaker. One patient required a subclavian covered stent implantation to treat a postimplant artery dissection associated with difficult surgical hemostasis. One patient was discharged in good condition but died of pneumonia 40 days after the procedure. All patients were asymptomatic on discharge, with good mid-term prosthesis performance.ConclusionsTranscatheter aortic valve implantation via a surgical subclavian approach seems safe and feasible, offering a new option to treat select, inoperable, and high-risk patients with severe aortic stenosis and peripheral vasculopathy

Human DPSCs fabricate vascularized woven bone tissue : a new tool in bone tissue engineering

Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells that have been successfully used in human bone tissue engineering. To establish whether these cells can lead to a bone tissue ready to be grafted, we checked DPSCs for their osteogenic and angiogenic differentiation capabilities with the specific aim of obtaining a new tool for bone transplantation. Therefore, hDPSCs were specifically selected from the stromal-vascular dental pulp fraction, using appropriate markers, and cultured. Growth curves, expression of bone-related markers, calcification and angiogenesis as well as an in vivo transplantation assay were performed. We found that hDPSCs proliferate, differentiate into osteoblasts and express high levels of angiogenic genes, such as vascular endothelial growth factor and platelet-derived growth factor A. Human DPSCs, after 40 days of culture, give rise to a 3D structure resembling a woven fibrous bone. These woven bone (WB) samples were analysed using classic histology and synchrotron-based, X-ray phase-contrast microtomography and holotomography. WB showed histological and attractive physical qualities of bone with few areas of mineralization and neovessels. Such WB, when transplanted into rats, was remodelled into vascularized bone tissue. Taken together, our data lead to the assumption that WB samples, fabricated by DPSCs, constitute a noteworthy tool and do not need the use of scaffolds, and therefore they are ready for customized regeneration

2-Pentadecyl-2-oxazoline ameliorates memory impairment and depression-like behaviour in neuropathic mice: possible role of adrenergic alpha2- and H3 histamine autoreceptors

Neuropathic pain (NP) remains an untreatable disease due to the complex pathophysiology that involves the whole pain neuraxis including the forebrain. Sensory dysfunctions such as allodynia and hyperalgesia are only part of the symptoms associated with neuropathic pain that extend to memory and affectivity deficits. The development of multi-target molecules might be a promising therapeutic strategy against the symptoms associated with NP. 2-pentadecyl-2-oxazoline (PEA-OXA) is a plant-derived agent, which has shown effectiveness against chronic pain and associated neuropsychiatric disorders. The molecular mechanisms by which PEA-OXA exerts its effects are, however, only partially known. In the current study, we show that PEA-OXA, besides being an alpha2 adrenergic receptor antagonist, also acts as a modulator at histamine H3 receptors, and report data on its effects on sensory, affective and cognitive symptoms associated with the spared nerve injury (SNI) model of neuropathic pain in mice. Treatment for 14 days with PEA-OXA after the onset of the symptoms associated with neuropathic pain resulted in the following effects: (i) allodynia was decreased; (ii) affective/cognitive impairment associated with SNI (depression, spatial, and working memories) was counteracted; (iii) long-term potentiation in vivo in the lateral entorhinal cortex-dentate gyrus (perforant pathway, LPP) was ameliorated, (iv) hippocampal glutamate, GABA, histamine, norepinephrine and dopamine level alterations after peripheral nerve injury were reversed, (v) expression level of the TH positive neurons in the Locus Coeruleus were normalized. Thus, a 16-day treatment with PEA-OXA alleviates the sensory, emotional, cognitive, electrophysiological and neurochemical alterations associated with SNI-induced neuropathic pain

Pentose phosphate pathway inhibition induce Endoplasmic Reticulum stress and autophagy

Pentose phosphate pathway (PPP) is a major glucose catabolism pathway that supplies the cell with a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) and ribose-5-phosphate. NADPH is necessary for the detoxification of reactive oxygen species (ROS) and reductive biosynthesis. A key player in this pathway is the enzyme glucose-6-phosphate dehydrogenase (G6PD) that reduces NADP+ to NADPH, oxidizes glucose-6-phosphate and prevents ROS accumulation. Here, we show that the natural molecule 3,4’,5-trihydroxystilbene-3-β-d-glucoside (Polydatin) inhibits glucose-6-phosphate dehydrogenase (G6PD). As expected, G6PD inhibition causes an imbalance in NADP+/NADPH ratio, leading to a redox imbalance, followed by Endoplasmic Reticulum (ER) stress, autophagy, cell cycle block and apoptosis. we have demonstrated a link between G6PD inhibition and ER stress, showing that Unfolded Protein Response mediator such as PERK and IRE-1 have a key role in inducing autophagy and apoptosis after PPP block. Moreover, combination of PPP inhibition with autophagy inhibitors, such as chloroquine, strongly potentiate cytotoxicity on cancer cells, evidencing the role of autophagy as an escaping mechanism. This results shows that double inhibition of PPP and autophagy may be an affective therapeutic strategy against cancer

Human DPSCs fabricate vascularized woven bone tissue: a new tool in bone tissue engineering

Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells that have been successfully used in human bone tissue engineering. To establish whether these cells can lead to a bone tissue ready to be grafted, we checked DPSCs for their osteogenic and angiogenic differentiation capabilities with the specific aim of obtaining a new tool for bone transplantation. Therefore, hDPSCs were specifically selected from the stromal-vascular dental pulp fraction, using appropriate markers, and cultured. Growth curves, expression of bone-related markers, calcification and angiogenesis as well as an in vivo transplantation assay were performed. We found that hDPSCs proliferate, differentiate into osteoblasts and express high levels of angiogenic genes, such as vascular endothelial growth factor and platelet-derived growth factor A. Human DPSCs, after 40 days of culture, give rise to a 3D structure resembling a woven fibrous bone. These woven bone (WB) samples were analysed using classic histology and synchrotron-based, X-ray phase-contrast microtomography and holotomography. WB showed histological and attractive physical qualities of bone with few areas of mineralization and neovessels. Such WB, when transplanted into rats, was remodelled into vascularized bone tissue. Taken together, our data lead to the assumption that WB samples, fabricated by DPSCs, constitute a noteworthy tool and do not need the use of scaffolds, and therefore they are ready for customized regeneration