Publications by authors named "Marta Columbaro"

59 Publications

Keratin nanoparticles and photodynamic therapy enhance the anticancer stem cells activity of salinomycin.

Mater Sci Eng C Mater Biol Appl 2021 Mar 23;122:111899. Epub 2021 Jan 23.

Department of Biology, University of Padova, Padova, Italy. Electronic address:

The high rates of aggressiveness, drug resistance and relapse of breast cancer (BC) are mainly attributed to the inability of conventional therapies to equally eradicate bulk differentiated cells and cancer stem cells (CSCs). To improve the effectiveness of BC treatments, we report the in-water synthesis of novel keratin-based nanoformulations, loaded with the CSC-specific drug salinomycin (SAL), the photosensitizer chlorin e6 (Ce6) and vitamin E acetate (SAL/Ce6@kVEs), which combine the capability of releasing SAL with the production of singlet oxygen upon light irradiation. In vitro experiments on BC cell lines and CSC-enriched mammospheres exposed to single or combined therapies showed that SAL/Ce6@kVEs determine synergistic cell killing, limit their self-renewal capacity and decrease the stemness potential by eradication of CSCs. In vivo experiments on zebrafish embryos confirmed the capacity of SAL nanoformulations to interfere with the Wnt/β-catenin signaling pathway, which is dysregulated in BC, thus identifying a target for further translation into pre-clinical models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.msec.2021.111899DOI Listing
March 2021

Exploring Metabolic Adaptations to the Acidic Microenvironment of Osteosarcoma Cells Unveils Sphingosine 1-Phosphate as a Valuable Therapeutic Target.

Cancers (Basel) 2021 Jan 15;13(2). Epub 2021 Jan 15.

Biomedical Science and Technology Lab, IRCCS Istituto Ortopedico Rizzoli, 40136 Bologna, Italy.

Acidity is a key player in cancer progression, modelling a microenvironment that prevents immune surveillance and enhances invasiveness, survival, and drug resistance. Here, we demonstrated in spheroids from osteosarcoma cell lines that the exposure to acidosis remarkably caused intracellular lipid droplets accumulation. Lipid accumulation was also detected in sarcoma tissues in close proximity to tumor area that express the acid-related biomarker LAMP2. Acid-induced lipid droplets-accumulation was not functional to a higher energetic request, but rather to cell survival. As a mechanism, we found increased levels of sphingomyelin and secretion of the sphingosine 1-phosphate, and the activation of the associated sphingolipid pathway and the non-canonical NF-ĸB pathway, respectively. Moreover, decreasing sphingosine 1-phosphate levels (S1P) by FTY720 (Fingolimod) impaired acid-induced tumor survival and migration. As a confirmation of the role of S1P in osteosarcoma, we found S1P high circulating levels (30.8 ± 2.5 nmol/mL, = 17) in the serum of patients. Finally, when we treated osteosarcoma xenografts with FTY720 combined with low-serine/glycine diet, both lipid accumulation (as measured by magnetic resonance imaging) and tumor growth were greatly inhibited. For the first time, this study profiles the lipidomic rearrangement of sarcomas under acidic conditions, suggesting the use of anti-S1P strategies in combination with standard chemotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers13020311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830496PMC
January 2021

Citrate Supplementation Restores the Impaired Mineralisation Resulting from the Acidic Microenvironment: An In Vitro Study.

Nutrients 2020 Dec 9;12(12). Epub 2020 Dec 9.

Biomedical Science and Technology Lab, IRCCS Istituto Ortopedico Rizzoli, via di Barbiano 1/10, 40136 Bologna, Italy.

Chronic metabolic acidosis leads to bone-remodelling disorders based on excessive mineral matrix resorption and inhibition of bone formation, but also affects the homeostasis of citrate, which is an essential player in maintaining the acid-base balance and in driving the mineralisation process. This study aimed to investigate the impact of acidosis on the osteogenic properties of bone-forming cells and the effects of citrate supplementation in restoring the osteogenic features impaired by the acidic milieu. For this purpose, human mesenchymal stromal cells were cultured in an osteogenic medium and the extracellular matrix mineralisation was analysed at the micro- and nano-level, both in neutral and acidic conditions and after treatment with calcium citrate and potassium citrate. The acidic milieu significantly decreased the citrate release and hindered the organisation of the extracellular matrix, but the citrate supplementation increased collagen production and, particularly calcium citrate, promoted the mineralisation process. Moreover, the positive effect of citrate supplementation was observed also in the physiological microenvironment. This in vitro study proves that the mineral matrix organisation is influenced by citrate availability in the microenvironment surrounding bone-forming cells, thus providing a biological basis for using citrate-based supplements in the management of bone-remodelling disorders related to chronic low-grade acidosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/nu12123779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763163PMC
December 2020

Benign albeit glycolytic: MCT4 expression and lactate release in giant cell tumour of bone.

Bone 2020 05 26;134:115302. Epub 2020 Feb 26.

Orthopaedic Pathophysiology and Regenerative Medicine Unit, IRCCS Istituto Ortopedico Rizzoli, Bologna, Italy; Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy.

Giant cell tumour of bone (GCTB) is a histologically benign, locally aggressive skeletal lesion with an unpredictable propensity to relapse after surgery and a rare metastatic potential. The microscopic picture of GCTB shows different cell types, including multinucleated giant cells, mononuclear cells of the macrophage-monocyte lineage, and spindle cells. The histogenesis of GCTB is still debated, and morphologic, radiographic or molecular features are not predictive of the clinical course. Characterization of the unexplored cell metabolism of GCTB offers significant clues for the understanding of this elusive pathologic entity. In this study we aimed to characterize GCTB energetic metabolism, with a particular focus on lactate release and the expression of monocarboxylate transporters, to lie down a novel path for understanding the pathophysiology of this tumour. We measured the expression of glycolytic markers (GAPDH, PKM2, MCT4, GLUT1, HK1, LDHA, lactate release) in 25 tissue samples of GCTB by immunostaining and by mRNA and ELISA analyses. We also evaluated MCT1 and MCT4 expression and oxidative markers (JC1 staining and Bec index) in tumour-derived spindle cell cultures and CD14+ monocytic cells. Finally, we quantified the intratumoural and circulating levels of lactate in a series of 17 subjects with GCTB. In sharp contrast to the benign histological features of GCTB, we found a high expression of glycolytic markers, with particular reference to MCT4. Unexpectedly, this was mainly confined to the giant cell, not proliferating cell component. Accordingly, GCTB patients showed higher levels of blood lactate as compared to healthy subjects. In conclusion, taken together, our data indicate that GCTB is characterized by a highly glycolytic metabolism of its giant cell component, opening new perspectives on the pathogenesis, the natural history, and the treatment of this lesion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bone.2020.115302DOI Listing
May 2020

Mesenchymal stromal cells mediated delivery of photoactive nanoparticles inhibits osteosarcoma growth in vitro and in a murine in vivo ectopic model.

J Exp Clin Cancer Res 2020 Feb 22;39(1):40. Epub 2020 Feb 22.

Unit of Orthopaedic Pathology and Osteoarticular Tissue Regeneration, IRCCS Istituto Ortopedico Rizzoli, Via di Barbiano 1/10, 40136, Bologna, Italy.

Background: Osteosarcoma (OS) is an aggressive malignant neoplasm that still suffers from poor prognosis in the case of distal metastases or occurrence of multi-drug resistance. It is therefore crucial to find novel therapeutic options able to go beyond these limitations and improve patients' survival. The objective of this study is to exploit the intrinsic properties of mesenchymal stromal cells (MSCs) to migrate and infiltrate the tumor stroma to specifically deliver therapeutic agents directly to cancer cells. In particular, we aimed to test the efficacy of the photoactivation of MSCs loaded with nanoparticles in vitro and in a murine in vivo ectopic osteosarcoma model.

Methods: AlPcS@FNPs were produced by adding tetra-sulfonated aluminum phthalocyanine (AlPcS) to an aqueous solution of positively charged poly-methyl methacrylate core-shell fluorescent nanoparticles (FNPs). The photodynamic therapy (PDT) effect is achieved by activation of the photosensitizer AlPcS in the near-infrared light with an LED source. Human MSCs were isolated from the bone marrow of five donors to account for inter-patients variability and used in this study after being evaluated for their clonogenicity, multipotency and immunophenotypic profile. MSC lines were then tested for the ability to internalize and retain the nanoparticles, along with their migratory properties in vitro. Photoactivation effect was evaluated both in a monolayer (2D) co-culture of AlPcS@FNPs loaded MSCs with human OS cells (SaOS-2) and in tridimensional (3D) multicellular spheroids (AlPcS@FNPs loaded MSCs with human OS cells, MG-63). Cell death was assessed by AnnexinV/PI and Live&Dead CalceinAM/EthD staining in 2D, while in the 3D co-culture, the cell killing effect was measured through ATP content, CalceinAM/EthD staining and TEM imaging. We also evaluated the effectiveness of AlPcS@FNPs loaded MSCs as delivery systems and the ability of the photodynamic treatment to kill cancer cells in a subcutaneous mouse model of OS by bioluminescence imaging (BLI) and histology.

Results: MSCs internalized AlPcS@FNPs without losing or altering their motility and viability in vitro. Photoactivation of AlPcS@FNPs loaded MSCs induced high level of OS cells death in the 2D co-culture. Similarly, in the 3D co-culture (MSCs:OS ratios 1:1 or 1:3), a substantial decrease of both MSCs and OS cells viability was observed. Notably, when increasing the MSCs:OS ratio to 1:7, photoactivation still caused more than 40% cells death. When tested in an in vivo ectopic OS model, AlPcS4@FNPs loaded MSCs were able to decrease OS growth by 68% after two cycles of photoactivation.

Conclusions: Our findings demonstrate that MSCs can deliver functional photosensitizer-decorated nanoparticles in vitro and in vivo and inhibit OS tumor growth. MSCs may be an effective platform for the targeted delivery of therapeutic nanodrugs in a clinical scenario, alone or in combination with other osteosarcoma treatment modalities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13046-020-01548-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036176PMC
February 2020

Loss of SMPD4 Causes a Developmental Disorder Characterized by Microcephaly and Congenital Arthrogryposis.

Am J Hum Genet 2019 10 5;105(4):689-705. Epub 2019 Sep 5.

Department of Clinical Genetics, ErasmusMC University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, the Netherlands. Electronic address:

Sphingomyelinases generate ceramide from sphingomyelin as a second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis. Children from 12 unrelated families presented with microcephaly, simplified gyral pattern of the cortex, hypomyelination, cerebellar hypoplasia, congenital arthrogryposis, and early fetal/postnatal demise. Genomic analysis revealed bi-allelic loss-of-function variants in SMPD4, coding for the neutral sphingomyelinase-3 (nSMase-3/SMPD4). Overexpression of human Myc-tagged SMPD4 showed localization both to the outer nuclear envelope and the ER and additionally revealed interactions with several nuclear pore complex proteins by proteomics analysis. Fibroblasts from affected individuals showed ER cisternae abnormalities, suspected for increased autophagy, and were more susceptible to apoptosis under stress conditions, while treatment with siSMPD4 caused delayed cell cycle progression. Our data show that SMPD4 links homeostasis of membrane sphingolipids to cell fate by regulating the cross-talk between the ER and the outer nuclear envelope, while its loss reveals a pathogenic mechanism in microcephaly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2019.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817560PMC
October 2019

Keratin nanoparticles co-delivering Docetaxel and Chlorin e6 promote synergic interaction between chemo- and photo-dynamic therapies.

J Photochem Photobiol B 2019 Oct 19;199:111598. Epub 2019 Aug 19.

Department of Biology, University of Padova, Via Ugo Bassi 58/B, 35100 Padova, Italy.

The combination of chemotherapy and photodynamic therapy (PDT) is considered a valuable strategy for increasing therapeutic response in cancer treatment, and the re-formulation of pharmaceuticals in biocompatible nanoparticles (NPs) is particularly appealing for the possibility of co-loading drugs exerting cytotoxicity by different mechanisms, with the aim to produce synergic effects. We report the in-water synthesis of a novel keratin-based nanoformulation for the co-delivery of the antimitotic Docetaxel (DTX) and the photosensitizer Chlorin e6 (Ce6). The drug-induced aggregation method allowed the formation of monodisperse NPs (DTX/Ce6-KNPs) with an average diameter of 133 nm and loaded with a drug ratio of 1:1.8 of Ce6 vs DTX. The efficacy of DTX/Ce6-KNPs was investigated in vitro in monolayers and spheroids of DTX-sensitive HeLa (HeLa-P) and DTX-resistant HeLa (HeLa-R) cells. In monolayers, the cytotoxic effects of DTX/Ce6-KNPs toward HeLa-P cells were comparable to those induced by free DTX + Ce6, while in HeLa-R cells the drug co-loading in KNPs produced synergic interaction between chemotherapy and PDT. Moreover, as respect to monotherapies, DTX/Ce6-KNPs induced stronger cytotoxicity to both HeLa-P and HeLa-R multicellular spheroids and reduced their volumes up to 50%. Overall, the results suggest that KNPs are very promising systems for the co-delivery of chemotherapeutics and PSs, favoring synergic interactions between PDT and chemotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jphotobiol.2019.111598DOI Listing
October 2019

Altered adipocyte differentiation and unbalanced autophagy in type 2 Familial Partial Lipodystrophy: an in vitro and in vivo study of adipose tissue browning.

Exp Mol Med 2019 08 2;51(8):1-17. Epub 2019 Aug 2.

CNR - National Research Council of Italy, Institute of Molecular Genetics "Luigi Luca Cavalli-Sforza", Unit of Bologna, Bologna, Italy.

Type-2 Familial Partial Lipodystrophy is caused by LMNA mutations. Patients gradually lose subcutaneous fat from the limbs, while they accumulate adipose tissue in the face and neck. Several studies have demonstrated that autophagy is involved in the regulation of adipocyte differentiation and the maintenance of the balance between white and brown adipose tissue. We identified deregulation of autophagy in laminopathic preadipocytes before induction of differentiation. Moreover, in differentiating white adipocyte precursors, we observed impairment of large lipid droplet formation, altered regulation of adipose tissue genes, and expression of the brown adipose tissue marker UCP1. Conversely, in lipodystrophic brown adipocyte precursors induced to differentiate, we noticed activation of autophagy, formation of enlarged lipid droplets typical of white adipocytes, and dysregulation of brown adipose tissue genes. In agreement with these in vitro results indicating conversion of FPLD2 brown preadipocytes toward the white lineage, adipose tissue from FPLD2 patient neck, an area of brown adipogenesis, showed a white phenotype reminiscent of its brown origin. Moreover, in vivo morpho-functional evaluation of fat depots in the neck area of three FPLD2 patients by PET/CT analysis with cold stimulation showed the absence of brown adipose tissue activity. These findings highlight a new pathogenetic mechanism leading to improper fat distribution in lamin A-linked lipodystrophies and show that both impaired white adipocyte turnover and failure of adipose tissue browning contribute to disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s12276-019-0289-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802660PMC
August 2019

Inducing cancer indolence by targeting mitochondrial Complex I is potentiated by blocking macrophage-mediated adaptive responses.

Nat Commun 2019 02 22;10(1):903. Epub 2019 Feb 22.

Dipartimento di Scienze Mediche e Chirurgiche, Università di Bologna, Via Massarenti 9, 40138, Bologna, Italy.

Converting carcinomas in benign oncocytomas has been suggested as a potential anti-cancer strategy. One of the oncocytoma hallmarks is the lack of respiratory complex I (CI). Here we use genetic ablation of this enzyme to induce indolence in two cancer types, and show this is reversed by allowing the stabilization of Hypoxia Inducible Factor-1 alpha (HIF-1α). We further show that on the long run CI-deficient tumors re-adapt to their inability to respond to hypoxia, concordantly with the persistence of human oncocytomas. We demonstrate that CI-deficient tumors survive and carry out angiogenesis, despite their inability to stabilize HIF-1α. Such adaptive response is mediated by tumor associated macrophages, whose blockage improves the effect of CI ablation. Additionally, the simultaneous pharmacological inhibition of CI function through metformin and macrophage infiltration through PLX-3397 impairs tumor growth in vivo in a synergistic manner, setting the basis for an efficient combinatorial adjuvant therapy in clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-08839-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385215PMC
February 2019

Functionalized Keratin as Nanotechnology-Based Drug Delivery System for the Pharmacological Treatment of Osteosarcoma.

Int J Mol Sci 2018 Nov 20;19(11). Epub 2018 Nov 20.

Institute of Organic Synthesis and Photoreactivity (ISOF), National Research Council (CNR), Via Gobetti, 101, 40129 Bologna, Italy.

Osteosarcoma therapy might be moving toward nanotechnology-based drug delivery systems to reduce the cytotoxicity of antineoplastic drugs and improve their pharmacokinetics. In this paper, we present, for the first time, an extensive chemical and in vitro characterization of dual-loaded photo- and chemo-active keratin nanoparticles as a novel drug delivery system to treat osteosarcoma. The nanoparticles are prepared from high molecular weight and hydrosoluble keratin, suitably functionalized with the photosensitizer Chlorin-e6 (Ce6) and then loaded with the chemotherapeutic drug Paclitaxel (PTX). This multi-modal PTX-Ce6@Ker nanoformulation is prepared by both drug-induced aggregation and desolvation methods, and a comprehensive physicochemical characterization is performed. PTX-Ce6@Ker efficacy is tested on osteosarcoma tumor cell lines, including chemo-resistant cells, using 2D and 3D model systems. The single and combined contributions of PTX and Ce6 is evaluated, and results show that PTX retains its activity while being vehiculated through keratin. Moreover, PTX and Ce6 act in an additive manner, demonstrating that the combination of the cytostatic blockage of PTX and the oxidative damage of ROS upon light irradiation have a far superior effect compared to singularly administered PTX or Ce6. Our findings provide the proof of principle for the development of a novel, nanotechnology-based drug delivery system for the treatment of osteosarcoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms19113670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6274803PMC
November 2018

Samp1 Mislocalization in Emery-Dreifuss Muscular Dystrophy.

Cells 2018 Oct 15;7(10). Epub 2018 Oct 15.

CNR Institute of Molecular Genetics, Unit of Bologna, 40136 Bologna, Italy.

linked-Emery-Dreifuss muscular dystrophy (EDMD2) is a rare disease characterized by muscle weakness, muscle wasting, and cardiomyopathy with conduction defects. The mutated protein lamin A/C binds several nuclear envelope components including the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and the inner nuclear membrane protein Samp1 (Spindle Associated Membrane Protein 1). Considering that Samp1 is upregulated during muscle cell differentiation and it is involved in nuclear movement, we hypothesized that it could be part of the protein platform formed by LINC proteins and prelamin A at the myotube nuclear envelope and, as previously demonstrated for those proteins, could be affected in EDMD2. Our results show that Samp1 is uniformly distributed at the nuclear periphery of normal human myotubes and committed myoblasts, but its anchorage at the nuclear poles is related to the presence of farnesylated prelamin A and it is disrupted by the loss of prelamin A farnesylation. Moreover, Samp1 is absent from the nuclear poles in EDMD2 myotubes, which shows that mutations associated with muscular dystrophy, due to reduced prelamin A levels in muscle cell nuclei, impair Samp1 anchorage. Conversely, SUN1 pathogenetic mutations do not alter Samp1 localization in myotubes, which suggests that Samp1 lies upstream of SUN1 in nuclear envelope protein complexes. The hypothesis that Samp1 is part of the protein platform that regulates microtubule nucleation from the myotube nuclear envelope in concert with pericentrin and LINC components warrants future investigation. As a whole, our data identify Samp1 as a new contributor to EDMD2 pathogenesis and our data are relevant to the understanding of nuclear clustering occurring in laminopathic muscle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells7100170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210792PMC
October 2018

Lamins and bone disorders: current understanding and perspectives.

Oncotarget 2018 Apr 27;9(32):22817-22831. Epub 2018 Apr 27.

CNR Institute of Molecular Genetics, Unit of Bologna, Bologna, Italy.

Lamin A/C is a major constituent of the nuclear lamina implicated in a number of genetic diseases, collectively known as laminopathies. The most severe forms of laminopathies feature, among other symptoms, congenital scoliosis, osteoporosis, osteolysis or delayed cranial ossification. Importantly, specific bone districts are typically affected in laminopathies. Spine is severely affected in LMNA-linked congenital muscular dystrophy. Mandible, terminal phalanges and clavicles undergo osteolytic processes in progeroid laminopathies and Restrictive Dermopathy, a lethal developmental laminopathy. This specificity suggests that lamin A/C regulates fine mechanisms of bone turnover, as supported by data showing that lamin A/C mutations activate non-canonical pathways of osteoclastogenesis, as the one dependent on TGF beta 2. Here, we review current knowledge on laminopathies affecting bone and LMNA involvement in bone turnover and highlight lamin-dependent mechanisms causing bone disorders. This knowledge can be exploited to identify new therapeutic approaches not only for laminopathies, but also for other rare diseases featuring bone abnormalities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.25071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5978267PMC
April 2018

Elevated TGF β2 serum levels in Emery-Dreifuss Muscular Dystrophy: Implications for myocyte and tenocyte differentiation and fibrogenic processes.

Nucleus 2018 01;9(1):292-304

j Institute of Molecular Genetics (IGM)-CNR, Unit of Bologna , Bologna , Italy.

Among rare diseases caused by mutations in LMNA gene, Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B are characterized by muscle weakness and wasting, joint contractures, cardiomyopathy with conduction system disorders. Circulating biomarkers for these pathologies have not been identified. Here, we analyzed the secretome of a cohort of patients affected by these muscular laminopathies in the attempt to identify a common signature. Multiplex cytokine assay showed that transforming growth factor beta 2 (TGF β2) and interleukin 17 serum levels are consistently elevated in the vast majority of examined patients, while interleukin 6 and basic fibroblast growth factor are altered in subgroups of patients. Levels of TGF β2 are also increased in fibroblast and myoblast cultures established from patient biopsies as well as in serum from mice bearing the H222P Lmna mutation causing Emery-Dreifuss Muscular Dystrophy in humans. Both patient serum and fibroblast conditioned media activated a TGF β2-dependent fibrogenic program in normal human myoblasts and tenocytes and inhibited myoblast differentiation. Consistent with these results, a TGF β2 neutralizing antibody avoided fibrogenic marker activation and myogenesis impairment. Cell intrinsic TGF β2-dependent mechanisms were also determined in laminopathic cells, where TGF β2 activated AKT/mTOR phosphorylation. These data show that TGF β2 contributes to the pathogenesis of Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B and can be considered a potential biomarker of those diseases. Further, the evidence of TGF β2 pathogenetic effects in tenocytes provides the first mechanistic insight into occurrence of joint contractures in muscular laminopathies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/19491034.2018.1467722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973167PMC
January 2018

Altered lipid metabolism could drive the bone marrow failure in fanconi anaemia.

Br J Haematol 2019 02 12;184(4):693-696. Epub 2018 Mar 12.

Haematology Unit, Istituto Giannina Gaslini, Genova, Italy.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bjh.15171DOI Listing
February 2019

Mandibuloacral dysplasia: A premature ageing disease with aspects of physiological ageing.

Ageing Res Rev 2018 Mar 5;42:1-13. Epub 2017 Dec 5.

CNR Institute of Molecular Genetics, Unit of Bologna, Via di Barbiano 1/10, I-40136, Bologna, Italy; Rizzoli Orthopedic Institute, Via di Barbiano 1/10, I-40136, Bologna, Italy. Electronic address:

Mandibuloacral dysplasia (MAD) is a rare genetic condition characterized by bone abnormalities including localized osteolysis and generalized osteoporosis, skin pigmentation, lipodystrophic signs and mildly accelerated ageing. The molecular defects associated with MAD are mutations in LMNA or ZMPSTE24 (FACE1) gene, causing type A or type B MAD, respectively. Downstream of LMNA or ZMPSTE24 mutations, the lamin A precursor, prelamin A, is accumulated in cells and affects chromatin dynamics and stress response. A new form of mandibuloacral dysplasia has been recently associated with mutations in POLD1 gene, encoding DNA polymerase delta, a major player in DNA replication. Of note, involvement of prelamin A in chromatin dynamics and recruitment of DNA repair factors has been also determined under physiological conditions, at the border between stress response and cellular senescence. Here, we review current knowledge on MAD clinical and pathogenetic aspects and highlight aspects typical of physiological ageing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.arr.2017.12.001DOI Listing
March 2018

Mesenchymal stem cells from preterm to term newborns undergo a significant switch from anaerobic glycolysis to the oxidative phosphorylation.

Cell Mol Life Sci 2018 03 3;75(5):889-903. Epub 2017 Oct 3.

Stem Cell Laboratory and Cell Therapy Center, IRCCS Istituto Giannina Gaslini, 16147, Genoa, Italy.

We evaluated the energy metabolism of human mesenchymal stem cells (MSC) isolated from umbilical cord (UC) of preterm (< 37 weeks of gestational age) and term (≥ 37 weeks of gestational age) newborns, using MSC from adult bone marrow as control. A metabolic switch has been observed around the 34th week of gestational age from a prevalently anaerobic glycolysis to the oxidative phosphorylation. This metabolic change is associated with the organization of mitochondria reticulum: preterm MSCs presented a scarcely organized mitochondrial reticulum and low expression of proteins involved in the mitochondrial fission/fusion, compared to term MSCs. These changes seem governed by the expression of CLUH, a cytosolic messenger RNA-binding protein involved in the mitochondria biogenesis and distribution inside the cell; in fact, CLUH silencing in term MSC determined a metabolic fingerprint similar to that of preterm MSC. Our study discloses novel information on the production of energy and mitochondrial organization and function, during the passage from fetal to adult life, providing useful information for the management of preterm birth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00018-017-2665-zDOI Listing
March 2018

Concentration-dependent metabolic effects of metformin in healthy and Fanconi anemia lymphoblast cells.

J Cell Physiol 2018 Feb 23;233(2):1736-1751. Epub 2017 Aug 23.

Hematology Unit, Istituto Giannina Gaslini, Genova, Italy.

Metformin (MET) is the drug of choice for patients with type 2 diabetes and has been proposed for use in cancer therapy and for treating other metabolic diseases. More than 14,000 studies have been published addressing the cellular mechanisms affected by MET. However, several in vitro studies have used concentrations of the drug 10-100-fold higher than the plasmatic concentration measured in patients. Here, we evaluated the biochemical, metabolic, and morphologic effects of various concentrations of MET. Moreover, we tested the effect of MET on Fanconi Anemia (FA) cells, a DNA repair genetic disease with defects in energetic and glucose metabolism, as well as on human promyelocytic leukemia (HL60) cell lines. We found that the response of wild-type cells to MET is concentration dependent. Low concentrations (15 and 150 µM) increase both oxidative phosphorylation and the oxidative stress response, acting on the AMPK/Sirt1 pathway, while the high concentration (1.5 mM) inhibits the respiratory chain, alters cell morphology, becoming toxic to the cells. In FA cells, MET was unable to correct the energetic/respiratory defect and did not improve the response to oxidative stress and DNA damage. By contrast, HL60 cells appear sensitive also at 150 μM. Our findings underline the importance of the MET concentration in evaluating the effect of this drug on cell metabolism and demonstrate that data obtained from in vitro experiments, that have used high concentrations of MET, cannot be readily translated into improving our understanding of the cellular effects of metformin when used in the clinical setting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jcp.26085DOI Listing
February 2018

The Role of Autophagy in the Maintenance of Stemness and Differentiation of Mesenchymal Stem Cells.

Stem Cell Rev Rep 2016 Dec;12(6):621-633

Orthopaedic Pathophysiology and Regenerative Medicine Unit, Istituto Ortopedico Rizzoli, Bologna, Italy.

Regulated self-consumption, also known as autophagy, is an evolutionary conserved process that degrades cellular components by directing them to the lysosomal compartment of eukaryotic cells. As a major intracellular degradation and recycling pathway, autophagy is crucial for maintaining and remodeling cellular homeostasis during normal cellular and tissue development. Recent studies have demonstrated that autophagy is necessary for the maintenance of cellular stemness and for a number of differentiation processes, including the lineage determination of mesenchymal stem cells. These are multipotent progenitor cells with self-renewal capacities that can give rise to a subset of tissues and thus hold a consistent potential in regenerative medicine. Here, we review the current literature on the complex liaison between autophagy induced by various extra- or intracellular stimuli and the molecular targets that affect mesenchymal stem cells proliferation and differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12015-016-9690-4DOI Listing
December 2016

Rapid and efficient magnetization of mesenchymal stem cells by dendrimer-functionalized magnetic nanoparticles.

Nanomedicine (Lond) 2016 06 18;11(12):1519-34. Epub 2016 May 18.

Cell & Gene Therapy, Department of Biomedicine, Basel University & Department of Surgery, Basel University Hospital, Basel, Switzerland.

Aim: Rapid and efficient magnetization of human bone marrow stromal cells (BMSC) through functionalized magnetic nanoparticles (MNP).

Methods: MNP were functionalized with poly(epsilon-lysine) dendrons exposing carboxybetaine residue (CB-MNP) to enhance binding to the cellular glycocalix. BMSC were incubated with CB-MNP or non-functionalized PAA-MNP for 5-30 min in suspension.

Results: CB-MNP functionalization increased the magnetization efficiency by threefold. Remarkably, 66% of cells were magnetized after only 5 min and the maximum efficiency of >80% was reached by 15 min. BMSC viability, proliferation and differentiation were not impaired: actually, adipogenic and osteogenic differentiation were even improved.

Conclusion: Carboxybetaine-dendron functionalization ensured rapid and efficient BMSC magnetization and allowed innovative suspension labeling, with a potential for bypassing adhesion culture of progenitors for regenerative medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2217/nnm-2016-0085DOI Listing
June 2016

Evaluation of energy metabolism and calcium homeostasis in cells affected by Shwachman-Diamond syndrome.

Sci Rep 2016 05 5;6:25441. Epub 2016 May 5.

Haematology Unit, Istituto Giannina Gaslini, 16148 Genova, Italy.

Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have ribosome biogenesis and their protein synthesis altered, which are two high-energy consuming cellular processes. The reported changes in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest an energy production defect in SDS cells. In our work, we have demonstrated that SDS cells display a Complex IV activity impairment, which causes an oxidative phosphorylation metabolism defect, with a consequent decrease in ATP production. These data were confirmed by an increased glycolytic rate, which compensated for the energetic stress. Moreover, the signalling pathways involved in glycolysis activation also appeared more activated; i.e. we reported AMP-activated protein kinase hyper-phosphorylation. Notably, we also observed an increase in a mammalian target of rapamycin phosphorylation and high intracellular calcium concentration levels ([Ca(2+)]i), which probably represent new biochemical equilibrium modulation in SDS cells. Finally, the SDS cell response to leucine (Leu) was investigated, suggesting its possible use as a therapeutic adjuvant to be tested in clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep25441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4857091PMC
May 2016

Lamin A/C sustains PcG protein architecture, maintaining transcriptional repression at target genes.

J Cell Biol 2015 Nov;211(3):533-51

Consiglio Nazionale delle Ricerche Institute of Cellular Biology and Neurobiology, Istituto di Ricovero e Cura a Carattere Scientifico Fondazione Santa Lucia, 00143 Rome, Italy

Beyond its role in providing structure to the nuclear envelope, lamin A/C is involved in transcriptional regulation. However, its cross talk with epigenetic factors--and how this cross talk influences physiological processes--is still unexplored. Key epigenetic regulators of development and differentiation are the Polycomb group (PcG) of proteins, organized in the nucleus as microscopically visible foci. Here, we show that lamin A/C is evolutionarily required for correct PcG protein nuclear compartmentalization. Confocal microscopy supported by new algorithms for image analysis reveals that lamin A/C knock-down leads to PcG protein foci disassembly and PcG protein dispersion. This causes detachment from chromatin and defects in PcG protein-mediated higher-order structures, thereby leading to impaired PcG protein repressive functions. Using myogenic differentiation as a model, we found that reduced levels of lamin A/C at the onset of differentiation led to an anticipation of the myogenic program because of an alteration of PcG protein-mediated transcriptional repression. Collectively, our results indicate that lamin A/C can modulate transcription through the regulation of PcG protein epigenetic factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201504035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4639869PMC
November 2015

All-trans retinoic acid and rapamycin normalize Hutchinson Gilford progeria fibroblast phenotype.

Oncotarget 2015 Oct;6(30):29914-28

National Research Council of Italy, Institute of Molecular Genetics, IGM-CNR, 40136 Bologna, Italy.

Hutchinson Gilford progeria syndrome is a fatal disorder characterized by accelerated aging, bone resorption and atherosclerosis, caused by a LMNA mutation which produces progerin, a mutant lamin A precursor. Progeria cells display progerin and prelamin A nuclear accumulation, altered histone methylation pattern, heterochromatin loss, increased DNA damage and cell cycle alterations. Since the LMNA promoter contains a retinoic acid responsive element, we investigated if all-trans retinoic acid administration could lower progerin levels in cultured fibroblasts. We also evaluated the effect of associating rapamycin, which induces autophagic degradation of progerin and prelamin A. We demonstrate that all-trans retinoic acid acts synergistically with low-dosage rapamycin reducing progerin and prelamin A, via transcriptional downregulation associated with protein degradation, and increasing the lamin A to progerin ratio. These effects rescue cell dynamics and cellular proliferation through recovery of DNA damage response factor PARP1 and chromatin-associated nuclear envelope proteins LAP2α and BAF. The combined all-trans retinoic acid-rapamycin treatment is dramatically efficient, highly reproducible, represents a promising new approach in Hutchinson-Gilford Progeria therapy and deserves investigation in ageing-associated disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.4939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4745772PMC
October 2015

Diverse lamin-dependent mechanisms interact to control chromatin dynamics. Focus on laminopathies.

Nucleus 2014 Sep-Oct;5(5):427-40

a CNR Institute for Molecular Genetics; Unit of Bologna and SC Laboratory of Musculoskeletal Cell Biology; Rizzoli Orthopedic Institute; Bologna, Italy.

Interconnected functional strategies govern chromatin dynamics in eukaryotic cells. In this context, A and B type lamins, the nuclear intermediate filaments, act on diverse platforms involved in tissue homeostasis. On the nuclear side, lamins elicit large scale or fine chromatin conformational changes, affect DNA damage response factors and transcription factor shuttling. On the cytoplasmic side, bridging-molecules, the LINC complex, associate with lamins to coordinate chromatin dynamics with cytoskeleton and extra-cellular signals.   Consistent with such a fine tuning, lamin mutations and/or defects in their expression or post-translational processing, as well as mutations in lamin partner genes, cause a heterogeneous group of diseases known as laminopathies. They include muscular dystrophies, cardiomyopathy, lipodystrophies, neuropathies, and progeroid syndromes. The study of chromatin dynamics under pathological conditions, which is summarized in this review, is shedding light on the complex and fascinating role of the nuclear lamina in chromatin regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/nucl.36289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4164485PMC
August 2015

Rapamycin treatment of Mandibuloacral dysplasia cells rescues localization of chromatin-associated proteins and cell cycle dynamics.

Aging (Albany NY) 2014 Sep;6(9):755-70

National Research Council of Italy, Institute of Molecular Genetics, IGM-CNR-IOR, Bologna, Italy. Rizzoli Orthopedic Institute, Laboratory of Musculoskeletal Cell Biology, Bologna, Italy.

Lamin A is a key component of the nuclear lamina produced through post-translational processing of its precursor known as prelamin A.LMNA mutations leading to farnesylated prelamin A accumulation are known to cause lipodystrophy, progeroid and developmental diseases, including Mandibuloacral dysplasia, a mild progeroid syndrome with partial lipodystrophy and altered bone turnover. Thus, degradation of prelamin A is expected to improve the disease phenotype. Here, we show different susceptibilities of prelamin A forms to proteolysis and further demonstrate that treatment with rapamycin efficiently and selectively triggers lysosomal degradation of farnesylated prelamin A, the most toxic processing intermediate. Importantly, rapamycin treatment of Mandibuloacral dysplasia cells, which feature very low levels of the NAD-dependent sirtuin SIRT-1 in the nuclear matrix, restores SIRT-1 localization and distribution of chromatin markers, elicits release of the transcription factor Oct-1 and determines shortening of the prolonged S-phase. These findings indicate the drug as a possible treatment for Mandibuloacral dysplasia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233654PMC
http://dx.doi.org/10.18632/aging.100680DOI Listing
September 2014

Muscular dystrophy-associated SUN1 and SUN2 variants disrupt nuclear-cytoskeletal connections and myonuclear organization.

PLoS Genet 2014 Sep 11;10(9):e1004605. Epub 2014 Sep 11.

Department of Biochemistry, University of Leicester, Leicester, United Kingdom.

Proteins of the nuclear envelope (NE) are associated with a range of inherited disorders, most commonly involving muscular dystrophy and cardiomyopathy, as exemplified by Emery-Dreifuss muscular dystrophy (EDMD). EDMD is both genetically and phenotypically variable, and some evidence of modifier genes has been reported. Six genes have so far been linked to EDMD, four encoding proteins associated with the LINC complex that connects the nucleus to the cytoskeleton. However, 50% of patients have no identifiable mutations in these genes. Using a candidate approach, we have identified putative disease-causing variants in the SUN1 and SUN2 genes, also encoding LINC complex components, in patients with EDMD and related myopathies. Our data also suggest that SUN1 and SUN2 can act as disease modifier genes in individuals with co-segregating mutations in other EDMD genes. Five SUN1/SUN2 variants examined impaired rearward nuclear repositioning in fibroblasts, confirming defective LINC complex function in nuclear-cytoskeletal coupling. Furthermore, myotubes from a patient carrying compound heterozygous SUN1 mutations displayed gross defects in myonuclear organization. This was accompanied by loss of recruitment of centrosomal marker, pericentrin, to the NE and impaired microtubule nucleation at the NE, events that are required for correct myonuclear arrangement. These defects were recapitulated in C2C12 myotubes expressing exogenous SUN1 variants, demonstrating a direct link between SUN1 mutation and impairment of nuclear-microtubule coupling and myonuclear positioning. Our findings strongly support an important role for SUN1 and SUN2 in muscle disease pathogenesis and support the hypothesis that defects in the LINC complex contribute to disease pathology through disruption of nuclear-microtubule association, resulting in defective myonuclear positioning.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1004605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161305PMC
September 2014

Inhibition of metalloproteinase activity in FANCA is linked to altered oxygen metabolism.

J Cell Physiol 2015 Mar;230(3):603-9

DIFAR-Biochemistry Lab., Department of Pharmacology, University of Genova, Genova, Italy.

Bone marrow (BM) failure, increased risk of myelodysplastic syndrome, acute leukaemia and solid tumors, endocrinopathies and congenital abnormalities are the major clinical problems in Fanconi anemia patients (FA). Chromosome instability and DNA repair defects are the cellular characteristics used for the clinical diagnosis. However, these biological defects are not sufficient to explain all the clinical phenotype of FA patients. The known defects are structural alteration in cell cytoskeleton, altered structural organization for intermediate filaments, nuclear lamina, and mitochondria. These are associated with different expression and/or maturation of the structural proteins vimentin, mitofilin, and lamin A/C suggesting the involvement of metalloproteinases (MPs). Matrix metalloproteinases (MMP) are involved in normal physiological processes such as human skeletal tissue development, maturation, and hematopoietic reconstitution after bone marrow suppression. Current observations upon the eventual role of MPs in FA cells are largely inconclusive. We evaluated the overall MPs activity in FA complementation group A (FANCA) cells by exposing them to the antioxidants N-acetyl cysteine (NAC) and resveratrol (RV). This work supports the hypothesis that treatment of Fanconi patients with antioxidants may be important in FA therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jcp.24778DOI Listing
March 2015

Treatment of FANCA cells with resveratrol and N-acetylcysteine: a comparative study.

PLoS One 2014 15;9(7):e104857. Epub 2014 Aug 15.

Hematology Unit, Istituto Giannina Gaslini, Genova, Italy.

Fanconi anemia (FA) is a genetic disorder characterised by chromosome instability, cytokine ipersensibility, bone marrow failure and abnormal haematopoiesis associated with acute myelogenous leukemia. Recent reports are contributing to characterize the peculiar FA metabolism. Central to these considerations appears that cells from complementation group A (FANCA) display an altered red-ox metabolism. Consequently the possibility to improve FA phenotypical conditions with antioxidants is considered. We have characterized from the structural and biochemical point of view the response of FANCA lymphocytes to N-acetyl-cysteine (NAC) and resveratrol (RV). Surprisingly both NAC and RV failed to revert all the characteristic of FA phenotype and moreover their effects are not super imposable. Our data suggest that we must be aware of the biological effects coming from antioxidant treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0104857PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134242PMC
November 2015

Lamins are rapamycin targets that impact human longevity: a study in centenarians.

J Cell Sci 2014 Jan 23;127(Pt 1):147-57. Epub 2013 Oct 23.

National Research Council of Italy, Institute of Molecular Genetics, Unit of Bologna IOR, 40136 Bologna, Italy.

The dynamic organisation of the cell nucleus is profoundly modified during growth, development and senescence as a result of changes in chromatin arrangement and gene transcription. A plethora of data suggests that the nuclear lamina is a key player in chromatin dynamics and argues in favour of a major involvement of prelamin A in fundamental mechanisms regulating cellular senescence and organism ageing. As the best model to analyse the role of prelamin A in normal ageing, we used cells from centenarian subjects. We show that prelamin A is accumulated in fibroblasts from centenarians owing to downregulation of its specific endoprotease ZMPSTE24, whereas other nuclear envelope constituents are mostly unaffected and cells do not enter senescence. Accumulation of prelamin A in nuclei of cells from centenarians elicits loss of heterochromatin, as well as recruitment of the inactive form of 53BP1, associated with rapid response to oxidative stress. These effects, including the prelamin-A-mediated increase of nuclear 53BP1, can be reproduced by rapamycin treatment of cells from younger individuals. These data identify prelamin A and 53BP1 as new targets of rapamycin that are associated with human longevity. We propose that the reported mechanisms safeguard healthy ageing in humans through adaptation of the nuclear environment to stress stimuli.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.133983DOI Listing
January 2014

Changes in vimentin, lamin A/C and mitofilin induce aberrant cell organization in fibroblasts from Fanconi anemia complementation group A (FA-A) patients.

Biochimie 2013 Oct 2;95(10):1838-47. Epub 2013 Jul 2.

IGM-CNR, Unit of Bologna c/o IOR, Bologna, Italy.

Growing number of publication has proved an increasing of cellular function of the Fanconi anemia proteins. To chromosome stability and DNA repair new roles have been attributed to FA proteins in oxidative stress response and homeostasis, immune response and cytokines sensibility, gene expression. Our work shows a new role for FA-A protein: the organization of the cellular structure. By 2D-PAGE of FA-A and correct fibroblasts treated and untreated with H2O2 we identify different expression of protein involved in the structural organization of nucleus, intermediate filaments and mitochondria. Immunofluorescence and electronic microscopy analysis clearly show an already altered cellular structure in normal culture condition and this worsted after oxidative stress. FA-A cell appears structurally prone to physiologic stress and this could explain part of the phenotype of FA cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biochi.2013.06.024DOI Listing
October 2013