RESUMO
In spite of the extensive research for developing new therapies, prostate cancer is still one of the major human diseases with poor prognosis and high mortality. Therefore, with the aim of identifying novel agents with antigrowth and pro-apoptotic activity on prostate cancer cells, in the present study, we evaluated the effect of lichen secondary metabolite physodic acid on cell growth in human prostate cancer cells. In addition, we tested the apoptotic activity of physodic acid on TRAIL-resistant LNCaP cells in combination with TRAIL. The cell viability was measured using MTT assay. LDH release, a marker of membrane breakdown, was also measured. For the detection of apoptosis, the evaluation of DNA fragmentation and caspase-3 activity assay were employed. The expression of proteins was detected by Western blot analysis. It was observed that physodic acid showed a dose-response relationship in the range of 12.5-50 µM concentrations in LNCaP and DU-145 cells, activating an apoptotic process. In addition, physodic acid sensitizes LNCaP cells to TRAIL-induced apoptosis. The combination of physodic acid with other anti-prostate cancer therapies could be considered a promising strategy that warrants further investigations.
Assuntos
Dibenzoxepinas , Neoplasias da Próstata , Apoptose , Linhagem Celular Tumoral , Dibenzoxepinas/farmacologia , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
Artemisia arborescens is an aromatic shrub whose essential oils are considered a potential source of molecules with industrial and pharmaceutical interest. The chemical profile of A. arborescens essential oils (EOs) was shown to be quite variable and various chemotypes have been identified. In this study, we compared the EOs composition of A. arborescens leaves and flowers collected from four different locations in Sicily. The EOs were assayed for their antiproliferative activity against A375 human malignant melanoma cells, also testing cell viability and cell membrane integrity. The evaluation of DNA fragmentation and caspase-3 activity assay was employed for the detection of apoptosis. The expression of Bcl-2, Bax, cleaved caspase-9, PTEN (Phosphatase and tensin homolog), Hsp70 (Heat Shock Protein 70 kilodaltons) and SOD (superoxide dismutase) proteins was evaluated by Western blot analysis. The levels of ROS and GSH were also analyzed. Results show that EOs presented significant differences in their composition, yield, and cytotoxic activity depending on the collection site. The chamazulene/camphor-rich EOs from plants collected in Acqua Calda (Lipari) resulted particularly active on melanoma cancer cells (IC50 values of 6.7 and 4.5 µg/mL), being able to trigger apoptotic death probably interfering with endogenous defense mechanisms. These oils may be considered as a natural resource of chamazulene, containing this compound up to 63%.
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Skin acts as a protective barrier between the body and the external environment. Skin wounds are a common inflammatory disorder for the solution of which plants and essential oils have been applied as a medical option for centuries. Origanum vulgare essential oil (OEO) is largely used in folk medicine, but its molecular mechanisms of action are not fully known. In this study, we evaluated the anti-inflammatory/antioxidant activity as well as wound healing capacity of a well-characterized OEO on human keratinocytes NCTC 2544 treated with interferon-gamma (IFN-γ) and histamine (H) or subjected to a scratch test. The expression of pro-inflammatory mediators such as reactive oxygen species (ROS), inter-cellular adhesion molecule (ICAM)-1, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 were verified. The DNA damage was shown by the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG) and activation of proliferating cell nuclear antigen (PCNA). Moreover, the abnormal modification of extracellular matrix components (ECM) was examined by determining matrix metalloproteinase (MMP)-1, and -12. Compared to untreated control, OEO showed efficacy in supporting and enhancing the cell motility. In IFN-γ and H treated cells, OEO displayed a significant reduction of ROS, ICAM-1, iNOS, COX-2, 8-OHdG, MMP-1, and MMP-12. OEO proved useful to treat inflammation and support cell motility during wound healing.
Assuntos
Anti-Inflamatórios/farmacologia , Queratinócitos/efeitos dos fármacos , Modelos Biológicos , Óleos Voláteis/farmacologia , Origanum/química , Cicatrização/efeitos dos fármacos , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , Ciclo-Oxigenase 2/metabolismo , Histamina/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Interferon gama/metabolismo , Queratinócitos/metabolismo , Metaloproteinases da Matriz/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The use of orchids in herbal medicine has a very long history. Dendrobium species are known to produce a variety of secondary metabolites such as phenanthrens, bibenzyls, fluorenones and sesquiterpenes, and alkaloids and are responsible for their wide variety of medicinal properties. For decades, bibenzyls, which are the main bioactive components derived from Dendrobium species, have been subjected to extensive investigation as likely candidates for cancer treatment. The present study was undertaken to investigate the effect of moscatilin, a bibenzyl derivative from the orchid Dendrobium loddigesii on human melanoma cells. In A375 cells compound moscatilin showed a clear dose-response relationship in the range of 6.25-50 µM concentrations. In addition, we demonstrated an apoptotic response after treatment of cancer cells with this bibenzyl compound at 6.25 and 12.5 µM concentrations that probably involves PTEN activity, inhibition of Hsp70 expression and reactive oxygen species production. Alternatively, the inhibition of the caspase cascade at higher concentrations, 25 and 50 µM, correlated with additional reactive oxygen species increase, probably switched the mode of moscatilin-induced cell death from apoptosis to necrosis.
Assuntos
Apoptose/efeitos dos fármacos , Compostos de Benzil/uso terapêutico , Dendrobium/química , Melanoma/tratamento farmacológico , Melanoma/patologia , Compostos de Benzil/química , Compostos de Benzil/farmacologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Glutationa/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Isocordin 1 and a series of 4-oxyalkyl-isocordoin analogues 2-8 were evaluated for their cytotoxicity effect against human melanoma cells (A2058). Analogues 4, 5, and 6 showed a higher inhibitory activity with IC50 values of 12.91 ± 0.031, 24.88 ± 0.013, and 11.62 ± 0.017, respectively. These analogues, 4, 5, and 6, also induced an apoptotic response at 12.5- and 25-µM concentrations. They inhibited the expression of antiapoptotic proteins Bcl-2 and Hsp70, a critical factor that promotes tumour cell survival. In contrast, Bax and caspase-9 expression, and caspase-3 enzyme resulted activated. These results were correlated to a DNA fragmentation typical of apoptosis and an increase of intracellular reactive oxygen species (ROS) levels. Alternatively, at higher concentration (50 µM), when the capacity of the cells to sustain Hsp70 synthesis is reduced, our results seem to indicate that necrosis was induced by a further increase in ROS production. Therefore, the central finding in the present study is that these molecules downregulates Hsp70 expression. Altogether, these results suggest that 4-oxyalkyl-isocordoin analogues 4, 5, and 6 deserve to be deeply investigated for a possible application as Hsp70 inhibitor in the management of melanoma.
Assuntos
Apoptose/efeitos dos fármacos , Catecóis/uso terapêutico , Proteínas de Choque Térmico HSP70/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Catecóis/farmacologia , HumanosRESUMO
Melanoma is a highly invasive cancer that resists most conventional treatments. Therefore, there is an urgent need to identify alternative anticancer agents able to affect new molecular targets. Drimys winteri (Winteraceae) is a medicinal plant, employed in Brazil and many countries, in folk medicine against a variety of ailments, especially for the treatment of fevers, ulcers, pains, affections of respiratory tract and cancers. Previous phytochemical studies have isolated and identified the presence of diverse classes of secondary metabolites in this plant such as sesquiterpenes. In an ongoing to identify new natural anticancer compounds for the treatment and/or prevention of melanoma, we study the effects of Drimys winteri bark ethyl acetate extract and its sesquiterpenes drimenol, nordrimenone, isonordrimenone and polygodial on human melanoma cells. The treatment of melanoma cells with extract, drimenol, isordrimenone and polygodial resulted in a significant reduction in cell viability. But, polygodial showed the highest inhibitory growth activity. In addition, we reported an apoptotic response after treatment with drimenol, isordrimenone and polygodial that probably involves the reduction of Hsp70 expression and reactive oxygen species production. Alternatively, the inhibition of caspase cascade at higher concentrations, correlated with additional reactive oxygen species increase, probably switches natural product-induced cell death from apoptosis to necrosis. Therefore, this evidence provides a scientific support for the anticancer employ of Drimys winteri in traditional medicinal and suggests that active molecules can be considered potential candidates to be tested also in in vivo models, alone or in combination with chemotherapy agents, for the management of melanoma.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Drimys/química , Extratos Vegetais/química , Antineoplásicos Fitogênicos/química , Caspase 3/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Drimys/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Melanoma/metabolismo , Melanoma/patologia , Casca de Planta/química , Casca de Planta/metabolismo , Extratos Vegetais/farmacologia , Plantas Medicinais/metabolismo , Sesquiterpenos Policíclicos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Terpenos/química , Terpenos/farmacologiaRESUMO
We investigated the complexation of celecoxib (CCB) into sulfobuthyl-ether-ß-cyclodextrin (SBE-ß-CD) for the realization of an inhalable dry-powder formulation containing gemcitabine (GEM) for lung anticancer therapy. Complexation increased the water solubility of CCB (0.003 mg/mL and 0.834 mg/mL for CCB free and complexed, respectively) and produced a quantitative dissolution of the drug within 15 min. The CCB/SBE-ß-CD inclusion complex showed a high stability constant (8131 M-1) not influenced by the presence of GEM in solution. Two-dimensional NMR experiments and computational studies demonstrated that the pyrazole ring of CCB penetrates deeper into SBE-ß-CD from the secondary rim. The aromatic rings are positioned at the edge of the cavity, establishing hydrogen bonds with the SBE-ß-CD that stabilized the complex. CCB showed limited cytotoxic activity on A549 cell lines. Complexation significantly increased activity passing from 30% to 45% cell mortality. Moreover, CCB/SBE-ß-CD strongly improved the cytotoxicity of GEM, observing about 60% of cell mortality for the combined formulation.
Assuntos
Antineoplásicos/farmacologia , Celecoxib/química , Desoxicitidina/análogos & derivados , beta-Ciclodextrinas/química , Células A549 , Antineoplásicos/química , Desoxicitidina/química , Desoxicitidina/farmacologia , Composição de Medicamentos , Sinergismo Farmacológico , Humanos , Simulação de Dinâmica Molecular , Pós , Solubilidade , Água/química , GencitabinaRESUMO
Melaleuca styphelioides, known as the prickly-leaf tea tree, contains a variety of bioactive compounds. The purposes of this study were to characterize the polyphenols extracted from Melaleuca styphelioides leaves and assess their potential antioxidant and anti-inflammatory effects. The polyphenol extracts were prepared by maceration with solvents of increasing polarity. The LC/MS-MS technique was used to identify and quantify the phenolic compounds. An assessment of the radical scavenging activity of all extracts was performed using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTSâº), and ferric reducing antioxidant power (FRAP) assays. The anti-inflammatory activity was determined on interferon gamma (IFN-γ)/histamine (H)-stimulated human NCTC 2544 keratinocytes by Western blot and RT-PCR. Compared to other solvents, methanolic extract presented the highest level of phenolic contents. The most frequent phenolic compounds were quercetin, followed by gallic acid and ellagic acid. DPPH, ABTSâº, and FRAP assays showed that methanolic extract exhibits strong concentration-dependent antioxidant activity. IFN-γ/H treatment of human NCTC 2544 keratinocytes induced the secretion of high levels of the pro-inflammatory mediator inter-cellular adhesion molecule-1 (ICAM-1), nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and nuclear factor kappa B (NF-κB), which were inhibited by extract. In conclusion, the extract of Melaleuca styphelioides leaves is rich in flavonoids, and presents antioxidant and anti-inflammatory proprieties. It can be proposed as a useful compound to treat inflammatory skin diseases.
Assuntos
Inflamação/tratamento farmacológico , Queratinócitos/efeitos dos fármacos , Melaleuca/química , Polifenóis/química , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Linhagem Celular , Flavonoides/química , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Histamina/toxicidade , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Interferon gama/toxicidade , Queratinócitos/patologia , Extratos Vegetais/química , Folhas de Planta/química , Polifenóis/isolamento & purificação , Polifenóis/farmacologiaRESUMO
The purpose of this research was to explore the behavior of aquaporins (AQPs) in an in vitro model of Parkinson's disease that is a recurrent neurodegenerative disorder caused by the gradual, progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Because of postmortem studies have provided evidences for oxidative damage and alteration of water flow and energy metabolism, we carried out an investigation about AQP4 and 9, demonstrated in the brain to maintain water and energy homeostasis. As an appropriate in vitro cell model, we used SH-SY5Y cultures and induced their differentiation into a mature dopaminergic neuron phenotype with retinoic acid (RA) alone or in association with phorbol-12-myristate-13-acetate (MPA). The association RA plus MPA provided the most complete and mature neuron phenotype, as demonstrated by high levels of ß-Tubulin III, MAP-2, and tyrosine hydroxylase. After validation of cell differentiation, the neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and H2O2 were applied to reproduce a Parkinson's-like stress. The results confirmed RA/MPA differentiated SH-SY5Y as a useful in vitro system for studying neurotoxicity and for using in a MPTP and H2O2-induced Parkinson's disease cell model. Moreover, the data demonstrated that neuronal differentiation, neurotoxicity, neuroinflammation, and oxidative stress are strongly correlated with dynamic changes of AQP4 and 9 transcription and transduction. New in vitro and in vivo experiments are needed to confirm these innovative outcomes.
Assuntos
Aquaporina 4 , Aquaporinas , Diferenciação Celular/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Doença de Parkinson/metabolismo , Aquaporina 4/efeitos dos fármacos , Aquaporina 4/metabolismo , Aquaporinas/efeitos dos fármacos , Aquaporinas/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/metabolismo , Humanos , Fármacos Neuroprotetores/farmacologia , Tretinoína/farmacologia , Tirosina 3-Mono-Oxigenase/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Prostate cancer is one of the most common forms of cancer in men, and research to find more effective and less toxic drugs has become necessary. In the frame of our ongoing program on traditionally used Salvia species from the Mediterranean Area, here we report the biological activities of Salvia aurea, S. judaica and S. viscosa essential oils against human prostate cancer cells (DU-145). The cell viability was measured by 3(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) test and lactate dehydrogenase (LDH) release was used to quantify necrosis cell death. Genomic DNA, caspase-3 activity, expression of cleaved caspase-9, B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X (Bax) proteins were analyzed in order to study the apoptotic process. The role of reactive oxygen species in cell death was also investigated. We found that the three essential oils, containing caryophyllene oxide as a main constituent, are capable of reducing the growth of human prostate cancer cells, activating an apoptotic process and increasing reactive oxygen species generation. These results suggest it could be profitable to further investigate the effects of these essential oils for their possible use as anticancer agents in prostate cancer, alone or in combination with chemotherapy agents.
Assuntos
Apoptose/efeitos dos fármacos , Medicamentos de Ervas Chinesas/administração & dosagem , Óleos Voláteis/administração & dosagem , Neoplasias da Próstata/tratamento farmacológico , Apoptose/genética , Canfanos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Medicamentos de Ervas Chinesas/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Óleos Voláteis/química , Panax notoginseng , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Espécies Reativas de Oxigênio/metabolismo , Salvia/química , Salvia miltiorrhiza , Proteína X Associada a bcl-2/genéticaRESUMO
Chondrocytes are cells of articular cartilage particularly sensitive to water transport and ionic and osmotic changes from extracellular environment and responsible for the production of the synovial fluid. Aquaporins (AQPs) are a family of water and small solute transport channel proteins identified in several tissues, involved in physiological pathways and in manifold human diseases. In a recent period, AQP1 and 3 seem to have a role in metabolic water regulation in articular cartilage of load bearing joints. The aim of this study was to examine the levels of AQP1 and 3 during the chondrogenic differentiation of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT). For the determination of chondrogenic markers and AQPs levels, glycosaminoglycans (GAGs) quantification, immunocytochemistry, RT-PCR, and Western blot were used after 0, 7, 14, 21, and 28 days from the start of differentiation. At 21 days, chondrocytes derived from AT-MSCs were able to produce augmented content of GAGs and significant quantity of SOX-9, lubricin, aggrecan, and collagen type II, suggesting hyaline cartilage formation, in combination with an increase of AQP3 and AQP1. However, while AQP1 level decreased after 21 days; AQP3 reached higher values at 28 days. The expression of AQP1 and 3 is a manifestation of physiological adaptation of functionally mature chondrocytes able to respond to the change of their internal environment influenced by extracellular matrix. The alteration or loss of expression of AQP1 and 3 could contribute to destruction of chondrocytes and to development of cartilage damage.
Assuntos
Aquaporina 1/metabolismo , Aquaporina 3/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Condrogênese , Células-Tronco Mesenquimais/metabolismo , Adulto , Agrecanas/genética , Agrecanas/metabolismo , Aquaporina 1/genética , Aquaporina 3/genética , Células Cultivadas , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Regulação da Expressão Gênica , Glicosaminoglicanos/metabolismo , Humanos , Cartilagem Hialina/metabolismo , Fenótipo , Proteoglicanas/genética , Proteoglicanas/metabolismo , Interferência de RNA , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Adulto JovemRESUMO
This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Authors. An anonymous reader made the authors aware of potential errors in the presentation and the experimental design for the Western blot data in Figure 3. Upon thorough investigation the authors concluded that in fact, in addition to an honest error (wrong image selected for inclusion into the article), the experimental design was not state-of-the-art in that the loading controls were run on parallel gels rather than on the gels to be probed for iNOS and collagen II. Therefore, in order to avoid any potentially wrong conclusions, the authors decided to retract the article, to confirm the data in a separate series of experiments and to submit the manuscript again after proper confirmation of the results and conclusions. The authors thank the anonymous reader, who spotted this error, and apologize for any inconvenience caused.
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Wilms' tumor gene 1 (WT1) plays complex roles in tumorigenesis, acting as tumor suppressor gene or an oncogene depending on the cellular context. A high WT1 expression level was described in various types of human bone and soft-tissue sarcomas, including osteosarcoma (OS), but its function in carcinogenesis is not yet well understood. This study investigated WT1 both in human OS tissues and in human OS MG-63 cell line in which WT1 gene is up-regulated. The results demonstrated that WT1 is expressed in 50% of human OS cases. WT1-silenced MG-63 cells showed deregulation of proteins of cell cycle and down-regulation of PI3K/AKT pathway. Induction of apoptotic programme was also established by activation of caspase-3 and increase of Bax/Bcl2 ratio and p53 protein. This study provided new findings on role of WT1 and indicated an association between WT1 expression, cell cycle and apoptotic machinery. In conclusion, WT1 acts as a tumour promoter in osteosarcoma and it could be a potential therapeutic target.
Assuntos
Apoptose/genética , Neoplasias Ósseas/patologia , Pontos de Checagem do Ciclo Celular/genética , Osteossarcoma/patologia , Proteínas WT1/biossíntese , Adolescente , Idoso , Western Blotting , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Criança , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes do Tumor de Wilms/fisiologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Osteossarcoma/genética , Transcriptoma , Proteínas WT1/genética , Adulto JovemRESUMO
Aquaporins (AQPs) are 13 integral membrane proteins that provide selective pores for the rapid movement of water and other uncharged solutes, across cell membranes. Recently, AQPs have been focused for their role in production, circulation, and homeostasis of the cerebrospinal fluid and their importance in several human diseases is becoming clear. This study investigated the time course (0, 14, and 28 days) of AQP1, 4, 7, 8, and 9 during the neural differentiation of human mesenchymal stem cells (MSCs) from adipose tissue (AT). For this purpose, two different media, enriched with serum or B-27 and N1 supplements, were applied to give a stimulus toward neural lineage. After 14 days, the cells were cultured with neuronal or glial differentiating medium for further 14 days. The results confirmed that AT-MSCs could be differentiated into neurons, astrocytes, and oligodendrocytes, expressing not only the typical neural markers but also specific AQPs depending on differentiated cell type. Our data demonstrated that at 28 days, AT-MSCs express only AQP1; astrocytes AQP1, 4, and 7; oligodendrocytes AQP1, 4, and 8; and finally neurons AQP1 and 7. This study provides fundamental insight into the biology of the mesenchymal stem cells and it suggests that AQPs can be potential neural markers.
Assuntos
Tecido Adiposo/metabolismo , Aquaporinas/biossíntese , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Tecido Adiposo/citologia , Adulto , Aquaporinas/genética , Células Cultivadas , Feminino , Expressão Gênica , Humanos , Masculino , Fenótipo , Adulto JovemRESUMO
The aim of this research was to assess the impact of a well-characterized extract from Citrus bergamia juice on adipogenesis and/or lipolysis using mesenchymal stem cells from human adipose tissue as a cell model. To evaluate the effects on adipogenesis, some cell cultures were treated with adipogenic medium plus 10 or 100 µg/mL of extract. To determine the properties on lipolysis, additional mesenchymal stem cells were cultured with adipogenic medium for 14 days and after this time added with Citrus bergamia for further 14 days. To verify adipogenic differentiation, oil red O staining at 7, 14, 21, and 28 days was performed. Moreover, the expression of peroxisome proliferator-activated receptor gamma (PPAR-γ), adipocytes fatty acid-binding protein (A-FABP), adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), monoglyceride lipase (MGL), 5'-adenosine monophosphate-activated protein kinase (AMPK)α1/2, and pAMPKα1/2 was evaluated by Western blot analysis and the release of glycerol by colorimetric assay. Citrus bergamia extract suppressed the accumulation of intracellular lipids in mesenchymal stem cells during adipogenic differentiation and promoted lipolysis by repressing the expression of adipogenic genes and activating lipolytic genes. Citrus bergamia extract could be a useful natural product for improving adipose mobilization in obesity-related disorders.
RESUMO
The first aim of the study was to identify the most appropriate time for differentiation of adipose tissue derived mesenchymal stem cells (MSCs) to chondrocytes, through the self-assembly process. For this purpose, the expression of some chondrocyte markers, such as collagen type I, collagen type II, RUNX2 and lubricin was investigated at different times (7, 14, 21 and 28 days) of chondrogenic differentiation of MSCs, by using immunohistochemistry and Western blot analysis. The second aim of the study was to demonstrate that the expression of lubricin, such as the expression of collagen type II, could be a possible biomarker for the detection of chondrocytes well-being and viability in the natural self-assembling constructs, called 'cell pellets'. Histology (hematoxylin and eosin) and histochemistry (alcian blue staining) methods were used to assess the chondrogenic differentiation of MSCs. The results showed that after 21 days the differentiated chondrocytes, when compared with MSCs cultured without chondrogenic medium (CD44, CD90 and CD105 positive; CD45, CD14 and CD34 negative), were able to produce significant quantities of collagen type I, collagen type II, and lubricin, suggesting hyaline cartilage formation. During the differentiation phase, the cells showed a reduced expression of RUNX2, a protein expressed by osteoblasts. Our studies demonstrated that 21 days is the optimum time for the implantation of chondrocytes differentiated from adipose tissue-derived MSCs. This information could be useful for the future development of cell-based repair therapies for degenerative diseases of articular cartilage.