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Teucrium chamaedrys L. is a typical European-Mediterranean species of the genus Teucrium. Among the phenolic compounds belonging to phenylethanoid glycosides (PGs), teucrioside (TS) is only found in this species, and it was previously demonstrated to be produced by in vitro-elicited cell cultures at levels higher than those found in leaves. However, T. chamaedrys cell suspension extracts (Cell-Ex) and pure TS have not been investigated yet for any biological effects. In this study, we evaluated the antioxidant and anti-melanogenesis activity of both Cell-Ex and TS in B16-F10 mouse melanoma cells. The results showed that Cell-Ex inhibited the reactive oxygen species formation evoked in B16-F10 cells by tert-butyl hydroperoxide and 5 J/cm2 of UVA, as well as the melanin increase stimulated by α-MSH or 20 J/cm2 of UVA. In parallel, a TS concentration equivalent to that present in Cell-Ex recorded the same biological effect profile, suggesting the main contribution of TS to the antioxidant and anti-melanogenic properties of Cell-Ex. Both Cell-Ex and TS also modulated the melanogenesis pathway through their ability to inhibit the tyrosinase activity both in a cell-free system and in B16-F10 cells stimulated by α-MSH. These results support the potential cosmeceutical use of Cell-Ex for protection against photooxidative damage and hyperpigmentation.
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Small-cell lung cancer (SCLC) is the most aggressive lung cancer type, and is associated with smoking, low survival rate due to high vascularization, metastasis and drug resistance. Alterations in MYC family members are biomarkers of poor prognosis for a large number of SCLC. In particular, MYCN alterations define SCLC cases with immunotherapy failure. MYCN has a highly restricted pattern of expression in normal cells and is an ideal target for cancer therapy but is undruggable by traditional approaches. We propose an innovative approach to MYCN inhibition by an MYCN-specific antigene-PNA oligonucleotide (BGA002)-as a new precision medicine for MYCN-related SCLC. We found that BGA002 profoundly and specifically inhibited MYCN expression in SCLC cells, leading to cell-growth inhibition and apoptosis, while also overcoming multidrug resistance. These effects are driven by mTOR pathway block in concomitance with autophagy reactivation, thus avoiding the side effects of targeting mTOR in healthy cells. Moreover, we identified an MYCN-related SCLC gene signature comprehending CNTFR, DLX5 and TNFAIP3, that was reverted by BGA002. Finally, systemic treatment with BGA002 significantly increased survival in MYCN-amplified SCLC mouse models, including in a multidrug-resistant model in which tumor vascularization was also eliminated. These findings warrant the clinical testing of BGA002 in MYCN-related SCLC.
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The most common pulmonary findings in opiate-related fatalities are congestion and oedema, as well as acute and/or chronic alveolar haemorrhage, the cause of which is thought to be a damage to the capillary endothelium related to ischemia. Human vascular mesenchymal stromal cells (vMSCs) play a fundamental role in tissue regeneration and repair after endothelial cell injury, and they express opioid receptors. The aim of this study was to assess the effect of in vitro morphine exposure on the physiological activity and maintenance of human vMSCs. vMSCs were obtained from abdominal aorta fragments collected during surgery repair and were exposed to incremental doses (0.1 mM, 0.4 mM, 0.8 mM and 1 mM) of morphine sulphate for 7 days. The effect was investigated through cell viability assessment, proliferation assay, reactive oxygen species (ROS) detection assay, senescence-associated ß-galactosidase assay, senescent-related markers (p21WAF1/CIP1 and p16INK4) and the apoptosis-related marker caspase 3. Moreover, an ultrastructural analysis by transmission electron microscopy and in vitro vascular differentiation were evaluated. Results showed a decrease of the cellular metabolic activity, a pro-oxidant and pro-senescence effect, an increase in intracellular ROS and the activation of the apoptosis signalling, as well as ultrastructural modifications and impairment of vascular differentiation after morphine treatment of vMSC. Although confirmation studies are required on real fatal opiate intoxications, the approach based on morphological and immunofluorescence methodologies may have a high potential also as a useful tool or as a complementary method in forensic pathology. The application of these techniques in the future may lead to the identification of new markers and morphological parameters useful as complementary investigations for drug-related deaths.
Assuntos
Células-Tronco Mesenquimais , Alcaloides Opiáceos , Humanos , Senescência Celular/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Alcaloides Opiáceos/farmacologia , Células-Tronco Mesenquimais/metabolismo , Derivados da Morfina/farmacologiaRESUMO
Osteoarthritis (OA) is described as a chronic degenerative disease characterized by the loss of articular cartilage. Senescence is a natural cellular response to stressors. Beneficial in certain conditions, the accumulation of senescent cells has been implicated in the pathophysiology of many diseases associated with aging. Recently, it has been demonstrated that mesenchymal stem/stromal cells isolated from OA patients contain many senescent cells that inhibit cartilage regeneration. However, the link between cellular senescence in MSCs and OA progression is still debated. In this study, we aim to characterize and compare synovial fluid MSCs (sf-MSCs), isolated from OA joints, with healthy sf-MSCs, investigating the senescence hallmarks and how this state could affect cartilage repair. Sf-MSCs were isolated from tibiotarsal joints of healthy and diseased horses with an established diagnosis of OA with an age ranging from 8 to 14 years. Cells were cultured in vitro and characterized for cell proliferation assay, cell cycle analysis, ROS detection assay, ultrastructure analysis, and the expression of senescent markers. To evaluate the influence of senescence on chondrogenic differentiation, OA sf-MSCs were stimulated in vitro for up to 21 days with chondrogenic factors, and the expression of chondrogenic markers was compared with healthy sf-MSCs. Our findings demonstrated the presence of senescent sf-MSCs in OA joints with impaired chondrogenic differentiation abilities, which could have a potential influence on OA progression.
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Células-Tronco Mesenquimais , Osteoartrite , Cavalos , Animais , Líquido Sinovial , Células Cultivadas , Osteoartrite/metabolismo , Senescência Celular/fisiologia , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , CondrogêneseRESUMO
Exfoliative cytology of human amniotic fluid (AF) has been extensively studied since 1940s, but no data exist in equine species. The AF compartment represents the environment in which the foetus grows and matures, and its composition changes, reflecting foetal well-being and development. The aim of this study was to describe for the first time the morphology of equine AF cells and amniotic membrane (AM) with light microscopy (LM) and transmission electron microscopy (TEM). AF was collected at parturition within 5 min after the appearance of the AM with a 60 mL syringe from 34 mares and samples of AM were collected from a subset of 7 mares with normal pregnancy hospitalized for attended parturition. For LM observation, a sample of cytocentrifuged fresh AF was stained with May-Grünwald Giemsa and AM sections were stained with H-E. For TEM observation, AF and AM were fixed, embedded in epoxy resins, then sectioned and stained with uranyl acetate and lead citrate solutions. Nucleated and anucleated squamous cells with basophilic cytoplasm, intensely basophilic cornified cells, polymorphonuclear cells, and clusters of eosinophilic amorphous substance were observed. Cells presumably derived from tracheal epithelium and small round nucleated cells with eosinophilic cytoplasm presumably derived from amniotic or urinary epithelium were occasionally found. Lamellar body-like structures (LBs) were present in some epithelial cells. In AM, epithelial, basal and mesenchymal layers were clearly visible with both techniques as previously described. Epithelial cells had several cytoplasmic vacuolization and microvilli were present on apical surface. The connective tissue presented fibroblasts, mesenchymal and rare polymorphonuclear cells, surrounded by abundant extracellular matrix, with distribution of collagen fibres. This is the first report about equine amniotic compartment description by LM and TEM. As recently reported in human medicine, the AM could be a second potential source of pulmonary surfactant, given the finding of LBs inside the cells which could have the same function as in humans. Further studies in samples collected at different gestational ages could increase the knowledge of AF cells and their modification during pregnancy, as well as a better comprehension of the role of AM as a secondary source of pulmonary surfactant in the horse. The diagnostic evaluation of AF cellular composition in high-risk pregnancies may also be investigated.
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Âmnio , Líquido Amniótico , Animais , Células Epiteliais , Feminino , Idade Gestacional , Cavalos , Parto , GravidezRESUMO
The discovery of the expression of opioid receptors in the skin and their role in orchestrating the process of tissue repair gave rise to questions regarding the potential effects of clinical morphine treatment in wound healing. Although short term treatment was reported to improve tissue regeneration, in vivo chronic administration was associated to an impairment of the physiological healing process and systemic fibrosis. Human mesenchymal stem cells (hMSCs) play a fundamental role in tissue regeneration. In this regard, acute morphine exposition was recently reported to impact negatively on the functional characteristics of hMSCs, but little is currently known about its long-term effects. To determine how a prolonged treatment could impair their functional characteristics, we exposed hMSCs to increasing morphine concentrations respectively for nine and eighteen days, evaluating in particular the fibrogenic potential exerted by the long-term exposition. Our results showed a time dependent cell viability decline, and conditions compatible with a cellular senescent state. Ultrastructural and protein expression analysis were indicative of increased autophagy, suggesting a relation to a detoxification activity. In addition, the enhanced transcription observed for the genes involved in the synthesis and regulation of type I collagen suggested the possibility that a prolonged morphine treatment might exert its fibrotic potential risk, even involving the hMSCs.
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Células-Tronco Mesenquimais/efeitos dos fármacos , Morfina/toxicidade , Cicatrização/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/análise , Colágeno Tipo I/metabolismo , Fibrose , Humanos , Células-Tronco Mesenquimais/fisiologia , Cultura Primária de Células , Testes de Toxicidade SubagudaRESUMO
Hydrogels based on short peptide molecules are interesting biomaterials with wide present and prospective use in biotechnologies. A well-known possible drawback of these materials can be their limited mechanical performance. In order to overcome this problem, we prepared Fmoc-Phe3self-assembling peptides by a biocatalytic approach, and we reinforced the hydrogel with graphene oxide nanosheets. The formulation here proposed confers to the hydrogel additional physicochemical properties without hampering peptide self-assembly. We investigated in depth the effect of nanocarbon morphology on hydrogel properties (i.e. morphology, viscoelastic properties, stiffness, resistance to an applied stress). In view of further developments towards possible clinical applications, we have preliminarily tested the biocompatibility of the composites. Our results showed that the innovative hydrogel composite formulation based on FmocPhe3 and GO is a biomaterial with improved mechanical properties that appears suitable for the development of biotechnological applications.
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Grafite , Hidrogéis , Peptídeos , Estudos ProspectivosRESUMO
Cellular senescence is a hallmark of ageing and it plays a key role in the development of age-related diseases. Abdominal aortic aneurysm (AAA) is an age related degenerative vascular disorder, characterized by a progressive dilatation of the vascular wall and high risk of rupture over time. Nowadays, no pharmacological therapies are available and the understanding of the molecular mechanisms that lead to AAA onset and development are poorly defined. In this study we investigated the cellular features of senescence in vascular mesenchymal stromal cells, isolated from pathological (AAA - MSCs) and healthy (h - MSCs) segments of human abdominal aorta and their implication in impairing the vascular repair ability of MSCs. Cell proliferation, ROS production, cell surface area, the expression of cyclin dependent kinase inhibitors p21CIP1 and p16INK4a, the activation of the DNA damage response and a dysregulated autophagy showed a senescent state in AAA - MSCs compared to h-MSCs. Moreover, a reduced ability to differentiate toward endothelial cells was observed in AAA - MSCs. All these data suggest that the accumulation of senescent vascular MSCs over time impairs their remodeling ability during ageing. This condition could support the onset and development of AAA.
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Aneurisma da Aorta Abdominal/metabolismo , Proliferação de Células , Senescência Celular , Células-Tronco Mesenquimais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Humanos , MasculinoRESUMO
Autosomal-dominant leukodystrophy (ADLD) is a rare fatal neurodegenerative disorder with overexpression of the nuclear lamina component, Lamin B1 due to LMNB1 gene duplication or deletions upstream of the gene. The molecular mechanisms responsible for driving the onset and development of this pathology are not clear yet. Vacuolar demyelination seems to be one of the most significant histopathological observations of ADLD. Considering the role of oligodendrocytes, astrocytes, and leukemia inhibitory factor (LIF)-activated signaling pathways in the myelination processes, this work aims to analyze the specific alterations in different cell populations from patients with LMNB1 duplications and engineered cellular models overexpressing Lamin B1 protein. Our results point out, for the first time, that astrocytes may be pivotal in the evolution of the disease. Indeed, cells from ADLD patients and astrocytes overexpressing LMNB1 show severe ultrastructural nuclear alterations, not present in oligodendrocytes overexpressing LMNB1. Moreover, the accumulation of Lamin B1 in astrocytes induces a reduction in LIF and in LIF-Receptor (LIF-R) levels with a consequential decrease in LIF secretion. Therefore, in both our cellular models, Jak/Stat3 and PI3K/Akt axes, downstream of LIF/LIF-R, are downregulated. Significantly, the administration of exogenous LIF can partially reverse the toxic effects induced by Lamin B1 accumulation with differences between astrocytes and oligodendrocytes, highlighting that LMNB1 overexpression drastically affects astrocytic function reducing their fundamental support to oligodendrocytes in the myelination process. In addition, inflammation has also been investigated, showing an increased activation in ADLD patients' cells.
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Astrócitos/metabolismo , Doenças Desmielinizantes/patologia , Lamina Tipo B/metabolismo , Transdução de Sinais , Astrócitos/citologia , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Células Cultivadas , Doenças Desmielinizantes/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Mediadores da Inflamação/metabolismo , Lamina Tipo B/genética , Fator Inibidor de Leucemia/metabolismo , Fator Inibidor de Leucemia/farmacologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Receptores de OSM-LIF/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Inflammatory bowel diseases (IBDs) are chronic and relapsing immune disorders that result, or possibly originate, from epithelial barrier defects. Intestinal organoids are a new reliable tool to investigate epithelial response in models of chronic inflammation. We produced organoids from the ulcerative colitis murine model Winnie to explore if the chronic inflammatory features observed in the parental intestine were preserved by the organoids. Furthermore, we investigated if quercetin administration to in vitro cultured organoids could suppress LPS-induced inflammation in wild-type organoids (WT-organoids) and spontaneous inflammation in ulcerative colitis organoids (UC-organoids). Our data demonstrate that small intestinal organoids obtained from Winnie mice retain the chronic intestinal inflammatory features characteristic of the parental tissue. Quercetin administration was able to suppress inflammation both in UC-organoids and in LPS-treated WT-organoids. Altogether, our data demonstrate that UC-organoids are a reliable experimental system for investigating chronic intestinal inflammation and pharmacological responses.
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Mucosa Intestinal/efeitos dos fármacos , Quercetina/farmacologia , Animais , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Mucosa Intestinal/ultraestrutura , Lipocalina-2/genética , Lipocalina-2/metabolismo , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Inibidor Secretado de Peptidases Leucocitárias/genética , Inibidor Secretado de Peptidases Leucocitárias/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVES: This study investigated the antitumor effect of a new nanomicellar complex obtained by combining the antitumor agent fenretinide with a quaternary amphiphilic amine RC16+ also endowed with antitumor activity. METHODS: The complex (Fen-RC16+) strongly improved the aqueous solubility of fenretinide (from 1,71 ± 0.08 µg/ml, pure fenretinide to 1500 ± 164 µg /ml, Fen-RC16+ complex) and provided a cytotoxic effect on SH-SY5Y neuroblastoma cell lines resulting from the intrinsic activity of both the complex components. Moreover, the mean size of the nanomicellar complex (ranging from 20 ± 1.97 nm to 40 ± 3.05 nm) was suitable for accumulation to the tumor site by the enhanced permeability and retention effect and the positive charge provided by the quaternary RC16+ induced adsorption of the complex on the tumor cell surface improving the intracellular concentration of fenretinide. RESULTS: All these characteristics made the Fen-RC16+ complex a multitasking system for antitumor therapy. CONCLUSION: Indeed its in vivo activity, evaluated on SH-SY5Y xenografts, was strong, and the tumor growth did not resume after the treatment withdrawal.
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Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Fenretinida/administração & dosagem , Nanoestruturas/administração & dosagem , Compostos de Amônio Quaternário/administração & dosagem , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Feminino , Fenretinida/química , Humanos , Camundongos Nus , Micelas , Neoplasias/tratamento farmacológico , Compostos de Amônio Quaternário/químicaRESUMO
Approximately half of high-risk neuroblastoma is characterized by MYCN amplification. N-Myc promotes tumor progression by inducing cell growth and inhibiting differentiation. MYCN has also been shown to play an active role in mitochondrial metabolism, but this relationship is not well understood. Although N-Myc is a known driver of the disease, it remains a target for which no therapeutic drug exists. Here, we evaluated a novel MYCN-specific antigene PNA oligonucleotide (BGA002) in MYCN-amplified (MNA) or MYCN-expressing neuroblastoma and investigated the mechanism of its antitumor activity. MYCN mRNA and cell viability were reduced in a broad set of neuroblastoma cell lines following BGA002 treatment. Furthermore, BGA002 decreased N-Myc protein levels and apoptosis in MNA neuroblastoma. Analysis of gene expression data from patients with neuroblastoma revealed that MYCN was associated with increased reactive oxygen species (ROS), downregulated mitophagy, and poor prognosis. Inhibition of MYCN caused profound mitochondrial damage in MNA neuroblastoma cells through downregulation of the mitochondrial molecular chaperone TRAP1, which subsequently increased ROS. Correspondingly, inhibition of MYCN reactivated mitophagy. Systemic administration of BGA002 downregulated N-Myc and TRAP1, with a concomitant decrease in MNA neuroblastoma xenograft tumor weight. In conclusion, this study highlights the role of N-Myc in blocking mitophagy in neuroblastoma and in conferring protection to ROS in mitochondria through upregulation of TRAP1. BGA002 is a potently improved MYCN-specific antigene oligonucleotide that reverts N-Myc-dysregulated mitochondrial pathways, leading to loss of the protective effect of N-Myc against mitochondrial ROS. SIGNIFICANCE: A second generation antigene peptide oligonucleotide targeting MYCN induces mitochondrial damage and inhibits growth of MYCN-amplified neuroblastoma cells.
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Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/patologia , Ácidos Nucleicos Peptídicos/farmacologia , Adolescente , Adulto , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Criança , Pré-Escolar , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactente , Recém-Nascido , Estimativa de Kaplan-Meier , Masculino , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitofagia/efeitos dos fármacos , Proteína Proto-Oncogênica N-Myc/antagonistas & inibidores , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/mortalidade , Ácidos Nucleicos Peptídicos/genética , Ácidos Nucleicos Peptídicos/uso terapêutico , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
Osteoarthritis is a degenerative disease that strongly correlates with age and promotes the breakdown of joint cartilage and subchondral bone. There has been a surge of interest in developing cell-based therapies, focused particularly on the use of mesenchymal stromal cells (MSCs) isolated from adult tissues. It seems that MSCs derived from synovial joint tissues exhibit superior chondrogenic ability, but their unclear distribution and low frequency actually limit their clinical application. To date, the influence of aging on synovial joint derived MSCs' biological characteristics and differentiation abilities remains unknown, and a full understanding of the mechanisms involved in cellular aging is lacking. The aim of this study was therefore to investigate the presence of age-related alterations in synovial fluid MSCs and their influence on the potential ability of MSCs to differentiate toward chondrogenic phenotypes. Synovial fluid MSCs, isolated from healthy equine donors from 3 to 40 years old, were cultured in vitro and stimulated towards chondrogenic differentiation for up to 21 days. An equine model was chosen due to the high degree of similarity of the anatomy of the knee joint to the human knee joint and as spontaneous disorders develop that are clinically relevant to similar human disorders. The results showed a reduction in cell proliferation correlated with age and the presence of age-related tetraploid cells. Ultrastructural analysis demonstrated the presence of morphological features correlated with aging such as endoplasmic reticulum stress, autophagy, and mitophagy. Alcian blue assay and real-time PCR data showed a reduction of efficiency in the chondrogenic differentiation of aged synovial fluid MSCs compared to young MSCs. All these data highlighted the influence of aging on MSCs' characteristics and ability to differentiate towards chondrogenic differentiation and emphasize the importance of considering age-related alterations of MSCs in clinical applications.
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Envelhecimento/patologia , Diferenciação Celular , Condrócitos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Osteoartrite/patologia , Envelhecimento/fisiologia , Animais , Proliferação de Células , Células Cultivadas , Senescência Celular/fisiologia , Condrócitos/patologia , Condrogênese/fisiologia , Modelos Animais de Doenças , Feminino , Cavalos , Humanos , Masculino , Células-Tronco Mesenquimais/patologia , Osteoartrite/fisiopatologia , Líquido Sinovial/citologiaRESUMO
Senescence can impair the therapeutic potential of stem cells. In this study, senescence-associated morphofunctional changes in periosteum-derived progenitor cells (PDPCs) from old and young individuals were investigated by combining cytofluorimetry, immunohistochemistry, and transmission electron microscopy. Cell cycle analysis demonstrated a large number of G0/G1 phase cells in PDPCs from old subjects and a progressive accumulation of G0/G1 cells during passaging in cultures from young subjects. Cytofluorimetry documented significant changes in light scattering parameters and closely correlated with the ultrastructural features, especially changes in mitochondrial shape and autophagy, which are consistent with the mitochondrial-lysosomal axis theory of ageing. The combined morphological, biofunctional, and ultrastructural approach enhanced the flow cytometric study of PDPC ageing. We speculate that impaired autophagy, documented in replicative senescent and old PDPCs, reflect a switch from quiescence to senescence. Its demonstration in a tissue with limited turnover-like the cambium layer of the periosteum, where reversible quiescence is the normal stem cell state throughout life-adds a new piece to the regenerative medicine jigsaw in an ageing society.
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Autofagia , Senescência Celular/fisiologia , Células-Tronco Mesenquimais , Periósteo/patologia , Adulto , Idoso de 80 Anos ou mais , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Células-Tronco Mesenquimais/fisiologia , Células-Tronco Mesenquimais/ultraestrutura , Microscopia Eletrônica de TransmissãoRESUMO
Wharton's jelly (WJ) is an important source of mesenchymal stem cells (MSCs) both in human and other animals. The aim of this study was to compare human and equine WJMSCs. Human and equine WJMSCs were isolated and cultured using the same protocols and culture media. Cells were characterized by analysing morphology, growth rate, migration and adhesion capability, immunophenotype, differentiation potential and ultrastructure. Results showed that human and equine WJMSCs have similar ultrastructural details connected with intense synthetic and metabolic activity, but differ in growth, migration, adhesion capability and differentiation potential. In fact, at the scratch assay and transwell migration assay, the migration ability of human WJMSCs was higher (P < 0.05) than that of equine cells, while the volume of spheroids obtained after 48 h of culture in hanging drop was larger than the volume of equine ones (P < 0.05), demonstrating a lower cell adhesion ability. This can also revealed in the lower doubling time of equine cells (3.5 ± 2.4 days) as compared to human (6.5 ± 4.3 days) (P < 0.05), and subsequently in the higher number of cell doubling after 44 days of culture observed for the equine (20.3 ± 1.7) as compared to human cells (8.7 ± 2.4) (P < 0.05), and to the higher (P < 0.05) ability to form fibroblast colonies at P3. Even if in both species tri-lineage differentiation was achieved, equine cells showed an higher chondrogenic and osteogenic differentiation ability (P < 0.05). Our findings indicate that, although the ultrastructure demonstrated a staminal phenotype in human and equine WJMSCs, they showed different properties reflecting the different sources of MSCs.
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Diferenciação Celular , Movimento Celular , Proliferação de Células , Células-Tronco Mesenquimais/citologia , Geleia de Wharton/citologia , Animais , Adesão Celular , Células Cultivadas , Condrogênese , Cavalos , Humanos , Células-Tronco Mesenquimais/ultraestrutura , Microscopia Eletrônica de Transmissão , Osteogênese , Especificidade da EspécieRESUMO
The lower incidence of ischemic heart disease in female with respect to male gender suggests the possibility that female sex hormones could have specific effects in cardiovascular protection. 17ß-Estradiol is the predominant premenopausal circulating form of estrogen and has a protective role on the cardiovascular system. Recent evidences suggest that gender can influence the response to cardiovascular medications; therefore, we hypothesized that sex hormones could also modulate the cardioprotective effects of nutraceutical compounds, such as the isothiocyanate sulforaphane, present in Brassica vegetables. This study was designed to explore the protective effects of sulforaphane in the presence of 17ß-estradiol against H2O2-induced oxidative stress in primary cultures of rat cardiomyocytes. Interestingly, 17ß-estradiol enhanced sulforaphane protective activity against H2O2-induced cell death with respect to sulforaphane or 17ß-estradiol alone as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide and lactate dehydrogenase assays. Moreover, 17ß-estradiol boosted sulforaphane ability to counteract oxidative stress, reducing intracellular reactive oxygen species and 8-hydroxy-2'-deoxyguanosine levels and increasing the expression of phase II enzymes. Using specific antagonists of estrogen receptor α and ß, we observed that these effects are not mediated by estrogen receptors. Otherwise, ERK1/2 and Akt signaling pathways seem to be involved, as the presence of specific inhibitors of these kinases reduced the protective effect of sulforaphane in the presence of 17ß-estradiol. Sulforaphane and 17ß-estradiol co-treatment counteracted cell morphology alterations induced by H2O2 as evidenced by transmission electron microscopy. Our results demonstrated, for the first time, that estrogens could enhance sulforaphane protective effects, suggesting that nutraceutical efficacy might be modulated by sex hormones.
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Cardiotônicos/farmacologia , Estradiol/farmacologia , Isotiocianatos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Sinergismo Farmacológico , Enzimas/genética , Enzimas/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Microscopia Eletrônica de Transmissão , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , SulfóxidosRESUMO
The tumor microenvironment contributes to cancer progression, in part through interactions between tumor and normal stromal cells. This study analyzed morphological and molecular changes induced in co-cultured human fibroblasts (HFs) and the MG-63 osteosarcoma cell line. Co-cultured cell monolayers were morphologically analyzed using high resolution scanning electron microscopy (HR-SEM), and trans-well assays were performed to assess cell migration and invasion. Proteins involved in inflammatory responses, cancer cell invasion, and angiogenesis were assessed using western blotting. HR-SEM showed progressive spatial orientation loss by fibroblasts in contact with MG-63s, while MG-63s proliferated rapidly and invaded HF space. Trans-well assays showed enhanced MG-63 migration in the presence of HFs. IL-6 expression was increased in co-cultured HFs, possibly stimulated by TNF-α. HFs do not normally express YKL-40 but did so in co-culture. Band densitometric analyses showed that increasing YKL-40 expression was followed by VEGF overexpression, especially in MG-63s. Finally, our results confirmed fibroblasts as the main matrix metalloproteinase source in this tumor microenvironment. Our study sheds new light on how tumor-stroma interactions promote tumor development and progression, and may support identification of novel anti-cancer therapeutics.
Assuntos
Neoplasias Ósseas/patologia , Fibroblastos Associados a Câncer/patologia , Movimento Celular , Osteossarcoma/patologia , Microambiente Tumoral , Western Blotting , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/ultraestrutura , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/ultraestrutura , Comunicação Celular , Linhagem Celular Tumoral , Proliferação de Células , Proteína 1 Semelhante à Quitinase-3/metabolismo , Técnicas de Cocultura , Progressão da Doença , Humanos , Interleucina-6/metabolismo , Metaloproteinases da Matriz/metabolismo , Microscopia Eletrônica de Varredura , Invasividade Neoplásica , Osteossarcoma/metabolismo , Osteossarcoma/ultraestrutura , Transdução de Sinais , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Intraductal papillary mucinous neoplasm (IPMN) is the most common cystic preneoplastic lesion of pancreatic cancer. We used an approach coupling high resolution cytogenetic analysis (Affymetrix Oncoscan FFPE Array) with clinically-oriented bioinformatic interpretation of data to understand the most relevant alterations of precursor lesions at different stages to identify new diagnostic markers. RESULTS: We identified multiple copy number alterations, particularly in lesions with severe dysplasia, with 7 IPMN with low-intermediate dysplasia carrying a nearly normal karyotype and 13 IPMN with complex Karyotype (> 4 alterations), showing high grade dysplasia. A specific gain of chromosome arm 3q was found in IPMN with complex Karyotype (92%). This gain of 3q is particularly interesting for the presence of oncogenes such as PIK3CA, GATA2 and TERC that are part of pathways that deregulate cell growth and promote disease progression. Quantitative PCR and FISH analysis confirmed the data . Further demonstration of the overexpression of the PIK3CA gene supports the identification of this alteration as a possible biomarker in the early identification of patients with IPMN at higher risk for disease progression. MATERIALS AND METHODS: High resolution cytogenetic analysis was performed in 20 formalin fixed paraffin embedded samples of IPMN by Oncoscan FFPE assay. Results were validated by qPCR and FISH analysis. CONCLUSIONS: The identification of these markers at an early stage of disease onset could help to identify patients at risk for cancer progression and new candidates for a more specific targeted therapy.
Assuntos
Adenocarcinoma Mucinoso/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Papilar/genética , Cromossomos Humanos Par 3 , Variações do Número de Cópias de DNA , Neoplasias Pancreáticas/genética , Adenocarcinoma Mucinoso/patologia , Biomarcadores Tumorais , Carcinoma Ductal Pancreático/patologia , Carcinoma Papilar/patologia , Análise Mutacional de DNA , Progressão da Doença , Humanos , Cariotipagem , Mutação , Gradação de Tumores , Neoplasias Pancreáticas/patologiaRESUMO
PURPOSE: We describe a novel class of antitumor amphiphilic amines (RCn) based on a tricyclic amine hydrophilic head and a hydrophobic linear alkyl tail of variable length. METHODS: We tested the lead compound, RC16, for cytotoxicity and mechanism of cell death in several cancer cell lines, anti tumor efficacy in mouse tumor models, and ability to encapsulate chemotherapy drugs. RESULTS: These compounds displayed strong cytotoxic activity against cell lines derived from both pediatric and adult cancers. The IC50 of the lead compound, RC16, for normal cells including human keratinocytes, human fibroblasts and human umbilical vein endothelial cells was tenfold higher than for tumor cells. RC16 exhibited significant antitumor effects in vivo using several human xenografts and a metastatic model of murine neuroblastoma by both intravenous and oral administration routes. The amphiphilic character of RC16 triggered a spontaneous molecular self-assembling in water with formation of micelles allowing complexation of Doxorubicin, Etoposide and Paclitaxel. These micelles significantly improved the in vitro antitumor activity of these drugs as the enhancement of their aqueous solubility also improved their biologic availability. CONCLUSIONS: RC16 and related amphiphilic amines may be useful as a novel cancer treatment.
Assuntos
Aminas/administração & dosagem , Antineoplásicos/administração & dosagem , Tensoativos/administração & dosagem , Aminas/química , Aminas/farmacocinética , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Doxorrubicina/farmacocinética , Portadores de Fármacos , Interações Medicamentosas , Etoposídeo/administração & dosagem , Etoposídeo/química , Etoposídeo/farmacocinética , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Nus , Micelas , Ácido N-Acetilneuramínico/química , Nanopartículas , Paclitaxel/administração & dosagem , Paclitaxel/química , Paclitaxel/farmacocinética , Tamanho da Partícula , Solubilidade , Propriedades de Superfície , Tensoativos/química , Tensoativos/farmacocinéticaRESUMO
BACKGROUND: The progression of malignant tumors does not depend exclusively on the autonomous properties of cancer cells; it is also influenced by tumor stroma reactivity and is under strict microenvironmental control. By themselves, stromal cells are not malignant, and they maintain normal tissue structure and function. However, through intercellular interactions or by paracrine secretions from cancer cells, normal stromal cells acquire abnormal phenotypes that sustain cancer cell growth and tumor progression. In their dysfunctional state, fibroblast and immune cells produce chemokines and growth factors that stimulate cancer cell growth and invasion. In our previous work, we established an in vitro model based on a monolayer co-culture system of healthy human fibroblasts (HFs) and human osteosarcoma cells (the MG-63 cell line) that simulates the microenvironment of tumor cells and healthy cells. The coexistence between MG-63 cells and HFs allowed us to identify the YKL-40 protein as the main marker for verifying the influence of tumor cells grown in contact with healthy cells. METHODS: In this study, we evaluated the interactions of HFs and MG-63 cells in a transwell co-culture system over 24 h, 48 h, 72 h, and 96 h. We analyzed the contributions of these populations to the tumor microenvironment during cancer progression, as measured by multiple markers. We examined the effect of siRNA knockdown of YKL-40 by tracking the subsequent changes in gene expression within the co-culture. We validated the expression of several genes, focusing on those involved in cancer cell invasion, inflammatory responses, and angiogenesis: TNF alpha, IL-6, MMP-1, MMP-9, and VEGF. We compared the results to those from a transwell co-culture without the YKL-40 knockdown. RESULTS: In a pro-inflammatory environment promoted by TNF alpha and IL-6, siRNA knockdown of YKL-40 caused a down-regulation of VEGF and MMP-1 expression in HFs. CONCLUSIONS: These findings demonstrated that the tumor microenvironment has an influence on the gene expression of healthy surrounding tissues and on the process of tumorigenicity and it is emerging as attractive targets for therapeutic strategies.