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1.
Cell Physiol Biochem ; 53(3): 480-495, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31486323

RESUMO

BACKGROUND/AIMS: Hypoxia Inducible Factor-1α (HIF-1α) is involved in cancer progression and is stabilized by the chaperone HSP90 (Heat Shock Protein 90), preventing degradation. Previously identified HSP90 inhibitors bind to the N-terminal pocket of HSP90, which blocks binding to HIF-1α and induces HIF-1α degradation. N-terminal inhibitors have failed in the clinic as single therapy treatments partially because they induce a heat shock response. SM molecules are HSP90 inhibitors that bind to the C-terminus of HSP90 and do not induce a heat shock response. The effects of these C-terminal inhibitors on HIF-1α are unreported. METHODS: HCT116, MDA-MB-231, PC3, and HEK293T cells were treated with HSP90 inhibitors. qRT-PCR and western blotting was performed to assess mRNA and protein levels of HIF-1α, HSP- and RACK1-related genes. siRNA was used to knockdown RACK1, while MG262 was used to inhibit proteasome activity. Dimethyloxalylglycine (DMOG) was used to inhibit activity of the prolyl hydroxylases (PHDs). Anti-angiogenic activity of HSP90 inhibitors was assessed using a HUVEC tubule formation assay. RESULTS: We show that SM compounds decrease HIF-1α target expression at the mRNA and protein level under hypoxia in colorectal, breast and prostate cancer cells, leading to cell death, without inducing a heat shock response. Surprisingly, we found that when the C-terminal of HSP90 is inhibited, HIF-1α degradation occurs through the proteasome and prolyl hydroxylases in an oxygen-dependent manner even in very low levels of oxygen (tumor hypoxia levels). RACK1 was not required for proteasomal degradation of HIF-1α. CONCLUSION: Our results suggest that by targeting the C-terminus of HSP90 we can exploit the prolyl hydroxylase and proteasome pathway to induce HIF-1α degradation in hypoxic tumors.


Assuntos
Hipóxia Celular/fisiologia , Proteínas de Choque Térmico HSP90/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Aminoácidos Dicarboxílicos/metabolismo , Western Blotting , Hipóxia Celular/genética , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células HCT116 , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células PC-3 , Prolil Hidroxilases/genética , Prolil Hidroxilases/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
2.
Isr Med Assoc J ; 21(7): 503, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31507132

RESUMO

BACKGROUND: Tumor treating fields (TTFields) are low-intensity, intermediate frequency electric fields that affect proliferating cells. TTFields are FDA approved for treatment of newly diagnosed and recurrent glioblastoma. Combining TTFields with immunotherapy is a rational approach due to their different mechanisms of action (MOA) and to the ability of TTFields to induce immunogenic cell death. Conversely, TTFields may interfere with immune functions critical for effective T-cell responses. OBJECTIVES: To evaluate the effects of TTFields on pivotal antitumoral T-cell functions. METHODS: T-cells from healthy donor peripheral blood (PB) or from viably dissociated human glioblastoma samples were cultured under normal or TTFields conditions, with or without superantigen stimulation. Multiparametric flow cytometry (8-color) was used to assess T-cell responses by monitoring select pivotal functions: proliferation (CFSE), IFNγ secretion, cytotoxic degranulation (CD107a), and activation/exhaustion (PD-1). Cellular viability was assessed in a dedicated assay. A chimeric antigen receptor (CAR) T-cell-based assay directly evaluated cellular cytotoxicity. RESULTS: Activated PB T-cells and tumor-infiltrating T-cells (TILs) preserved all monitored anti- tumoral functions under TTFields, apart from proliferation. This finding also applied specifically to PD-1 + TILs, comprised predominantly of tumor antigen-specific cells. Activated T-cells that attempted to proliferate under TTFields demonstrated decreased viability, in line with TTField MOA. Small or no reduction in viability was found in T-cells that did not attempt to proliferate, whether activated or resting. CONCLUSIONS: All monitored anti-tumoral T cell functions, except for proliferation, were unhindered by TTFields. Our results support further investigation into combinations of TTFields with T-cell based immunotherapeutic approaches.


Assuntos
Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Terapia por Estimulação Elétrica/métodos , Glioblastoma/terapia , Citometria de Fluxo/métodos , Glioblastoma/patologia , Humanos , Recidiva Local de Neoplasia , Linfócitos T/citologia
3.
Life Sci ; 232: 116665, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31323273

RESUMO

AIMS: Overexpression of the mechanistic target of rapamycin (mTOR), a member of the PIKK (phosphoinositide kinase-related kinase) family, protects cardiomyocytes from cell death induced by pathological stimuli such as ischemia. We previously reported that posttranslational modification of mTOR plays an important role in regulating cardiac mTOR expression. The aim of this study was to see if Tel2 (telomere maintenance 2), a protein that regulates the abundance of PIKKs, confers similar cardioprotective effects as mTOR. Tel2 is not well-characterized in cardiomyocytes, therefore we examined the effects of Tel2 on cardiomyocyte viability under ischemic stress conditions. MATERIALS AND METHODS: We overexpressed Tel2 or silenced Tel2 with siRNA in the HL-1 cardiomyocyte cell line to survey the effects of Tel2 overexpression and downregulation on cell survival during hypoxia. Adult mouse cardiomyocytes transfected with Tel2 adenoviruses were used to test whether Tel2 sufficiently prevented cardiomyocyte cell death against hydrogen peroxide (H2O2). KEY FINDINGS: Overexpressing Tel2 increased mTOR expression with a concomitant increase in mTOR Complex 1 (mTORC1) and mTORC2 activity in HL-1 cells. Tel2 deletion decreased mTOR expression, and mTORC1 and mTORC2 activity accordingly. In both HL-1 cells and adult mouse cardiomyocytes, Tel2 overexpression protected cardiomyocytes under ischemic stress. These effects were mTOR-dependent, as mTOR inhibitors blunted the effects of Tel2. While gene silencing of Tel2 did not affect cell survival under normoxia, Tel2 silencing made cardiomyocytes more vulnerable to cell death under hypoxia. SIGNIFICANCE: Upregulating Tel2 expression increases mTOR-mediated cardiomyocyte survival and targeting Tel2 could be another therapeutic strategy against ischemic heart disease.


Assuntos
Sobrevivência Celular/fisiologia , Miócitos Cardíacos/citologia , Proteínas de Ligação a Telômeros/fisiologia , Adenoviridae/genética , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Inativação Gênica , Peróxido de Hidrogênio/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , Transdução de Sinais , Proteínas de Ligação a Telômeros/genética , Transfecção
4.
Mol Carcinog ; 58(9): 1640-1647, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31264291

RESUMO

T-cell protein tyrosine phosphatase (TC-PTP, encoded by PTPN2) is a nonreceptor PTP that is most highly expressed in hematopoietic tissues. TC-PTP modulates a variety of physiological functions including cell cycle progression, cell survival and proliferation, and hematopoiesis through tyrosine dephosphorylation of its target substrates, such as EGFR, JAK1, JAK3, STAT1, and STAT3. Studies with whole or tissue-specific loss of TC-PTP function transgenic mice have shown that TC-PTP has crucial roles in the regulation of the immune response, insulin signaling, and oncogenic signaling. More recently, the generation of epidermal-specific TC-PTP-deficient mice for use in multistage skin carcinogenesis bioassays demonstrated that TC-PTP suppresses skin tumor formation by negatively regulating STAT3 and AKT signaling. Further investigation showed that TC-PTP also minimizes UVB-induced epidermal cell damage by promoting apoptosis through the negative regulation of Flk-1/JNK signaling. These findings provide major evidence for a tumor suppressive function for TC-PTP against environment-induced skin cancer. Here, we will discuss TC-PTP, its substrates, and its functions with an emphasis on its role in skin carcinogenesis.


Assuntos
Carcinogênese/metabolismo , Células Epiteliais/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Animais , Ciclo Celular/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Epiderme/metabolismo , Epiderme/fisiologia , Células Epiteliais/fisiologia , Hematopoese/fisiologia , Humanos , Transdução de Sinais/fisiologia
5.
Life Sci ; 232: 116601, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31252000

RESUMO

AIMS: Tet1, Tet2, and interleukin-6 (IL-6) have been linked to atherosclerosis. Whether Tet3 has a relationship with atherosclerosis and IL-6 was unknown. This study aims to determine the link between Tet3 and IL-6, and the role of Tet3 in prenatal hypoxia-induced atherosclerosis in offspring rats. MAIN METHODS: Pregnant rats were divided into hypoxia and control group. Their male offspring were tested at 20 months old. Hematoxylin-eosin staining and transmission electron microscopic staining were used. Gene mRNA and protein levels were measured with q-PCR or Western blotting. Cell viability and migration was tested with MTT or cell scratch assay. 5-hmC and 5-mC expression were obtained by qGlucMS-PCR; 5-hmC and 5-mC activity were obtained by dot blotting. KEY FINDINGS: Chronic prenatal hypoxia increased Tet3 and IL-6 expression, and decreased Tet3 activity in offspring rats. GlucMS-qPCR showed the percentage of 5-hmC was significantly up-regulated in the promoter of IL-6 in both the rats and cells. Moreover, 5-hmC percentage also was increased in the A7r5 cells transfected with Tet3. Furthermore, Tet3 promoted proliferation and migration of A7r5 cells. However, Tet3 was not sensitive to acute hypoxia, while influenced by HIF-1α DNA element. SIGNIFICANCE: Tet3 enhanced IL-6 expression though up-regulating 5-hmC percentage in the IL-6 promoter.


Assuntos
Aterosclerose/metabolismo , Desoxicitidina/análogos & derivados , Dioxigenases/metabolismo , Hipóxia/metabolismo , Interleucina-6/biossíntese , Animais , Aterosclerose/genética , Aterosclerose/patologia , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Desoxicitidina/metabolismo , Epigênese Genética , Feminino , Hipóxia/genética , Hipóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Oxigenases de Função Mista/metabolismo , Gravidez , Complicações na Gravidez/metabolismo , Complicações na Gravidez/patologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ativação Transcricional , Regulação para Cima
6.
Nat Rev Gastroenterol Hepatol ; 16(9): 544-558, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31253940

RESUMO

Telomerase is a key enzyme for cell survival that prevents telomere shortening and the subsequent cellular senescence that is observed after many rounds of cell division. In contrast, inactivation of telomerase is observed in most cells of the adult liver. Absence of telomerase activity and shortening of telomeres has been implicated in hepatocyte senescence and the development of cirrhosis, a chronic liver disease that can lead to hepatocellular carcinoma (HCC) development. During hepatocarcinogenesis, telomerase reactivation is required to enable the uncontrolled cell proliferation that leads to malignant transformation and HCC development. Part of the telomerase complex, telomerase reverse transcriptase, is encoded by TERT, and several mechanisms of telomerase reactivation have been described in HCC that include somatic TERT promoter mutations, TERT amplification, TERT translocation and viral insertion into the TERT gene. An understanding of the role of telomeres and telomerase in HCC development is important to develop future targeted therapies and improve survival of this disease. In this Review, the roles of telomeres and telomerase in liver carcinogenesis are discussed, in addition to their potential translation to clinical practice as biomarkers and therapeutic targets.


Assuntos
Carcinoma Hepatocelular/metabolismo , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Humanos , Fígado/metabolismo , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Camundongos , Mutação , Telomerase/genética , Telômero/genética
7.
Life Sci ; 232: 116597, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31238052

RESUMO

LncRNA SNHG3 (SNHG3) is involved in tumor development and progression, but little is known about how SNHG3 functions in laryngeal carcinoma (LC). Real time-PCR (RT-PCR) was used to estimate the expression of SNHG3 in LC tissues and cell lines TU212, TU686, and Hep-2. Cell viability, migration, and invasion were evaluated. Our results showed increased SNHG3 in LC tissues and cell lines. Loss of function of SNHG3 reduced cell viability, migration, and invasion of TU212 and TU686 cells. Western blot analyses demonstrated that the protein levels of MMP2 and MMP9 decreased after SNHG3 silencing. Additionally, bioinformatics software predicted that SNHG3 could sponge miR-384 at the 3'-UTR with complementary binding sites, which was validated by a dual-luciferase reporter assay. RT-PCR analysis revealed that knockdown of SNHG3 upregulated miR-384 expression and that overexpression of miR-384 decreased SNHG3. Furthermore, a dual-luciferase reporter assay showed that miR-384 could bind to the 3'-UTR of WEE1, and inhibition of miR-384 markedly increased WEE1 expression. The mRNA and protein levels of WEE1 were downregulated upon deletion of SNGH3. Suppression of WEE1 partly abolished the tumorigenic migration and invasion potential of the miR-384 inhibitor in migration and invasion. Inhibition of miR-384 partially reversed the biological activities of SNHG3 in TU212 and TU686 cells. Collectively, our results indicate that SNHG3 regulated LC cell migration and invasion via the miR-384/WEE1 axis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/metabolismo , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Regiões 3' não Traduzidas , Carcinogênese , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Humanos , Neoplasias Laríngeas/patologia , MicroRNAs/genética , Invasividade Neoplásica , Proteínas Nucleares/genética , Proteínas Tirosina Quinases/genética
8.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1122-1123: 83-89, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31173996

RESUMO

For magnetic control of cells for biomedical applications such as targeting of immune cells to tumors, cells must be magnetizable. For that, cells are incubated with superparamagnetic iron oxide nanoparticles (SPIONs) to take them up and thus become magnetizable. When using adherent cells, non-ingested SPIONs can be easily removed by rinsing of the particles regardless of their colloidal stability in cell culture medium. However, if suspension cells such as T cells are to be loaded with SPIONs, established methods to separate excess nanoparticles from cells are based on physicochemical parameters such as density, size or magnetizability. Thus, colloidal stability of the particles is of great importance, since only colloidally stable SPIONs can be completely separated from the cells due to their physicochemical differences. Aggregates of colloidally meta- or unstable particles cannot, however, be separated from cells due to their overlapping sizes and densities. Thus, development of an alternative method for the separation of nanoparticle aggregates from suspension cells is urgently needed. Here, we present an affinity chromatographic separation method based on immunohistochemical properties of the respective cells. A desthiobiotinylated antibody against a cellular surface antigen (here CD90.2 receptor on EL4 T cells) is immobilized on a streptavidin agarose column optimized for cell purification. Subsequently the column is loaded with the particle/cell suspension so that the cells bind to the column. After removing the particles by washing, the cells can be gently eluted with biotin solution under physiological conditions. This allows >95% of the excess iron concentration to be removed while maintaining cell viability.


Assuntos
Cromatografia de Afinidade/métodos , Separação Imunomagnética/métodos , Nanopartículas de Magnetita/química , Animais , Anticorpos/metabolismo , Biotina/química , Linhagem Celular , Sobrevivência Celular/fisiologia , Coloides/química , Camundongos , Estreptavidina/química
9.
Int J Oncol ; 55(1): 81-92, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31180521

RESUMO

Renal cell carcinoma (RCC) is the most common type of kidney cancer. By analysing The Cancer Genome Atlas (TCGA) database, 16 genes were identified to be consistently highly expressed in RCC tissues compared with the matched para­tumour tissues. Using a high­throughput cell viability screening method, it was found that downregulation of only two genes significantly inhibited the viability of 786­O cells. Among the two genes, pleckstrin homology domain containing O1 (PLEKHO1) has never been studied in RCC, to the best of our knowledge, and its expression level was shown to be associated with the prognosis of patients with RCC in TCGA dataset. The upregulation of PLEKHO1 in RCC was first confirmed in 30 paired tumour and para­tumour tissues. Then, the effect of PLEKHO1 on cell proliferation and apoptosis was assessed in vitro. Additionally, xenograft tumour models were established to investigate the function of PLEKHO1 in vivo. The results showed that PLEKHO1 knockdown significantly inhibited cell viability and facilitated apoptosis in vitro and impaired tumour formation in vivo. Thus, PLEKHO1 is likely to be associated with the viability of RCC cells in vitro and in vivo. Further gene expression microarray and co­expression analyses showed that PLEKHO1 may be involved in the serine/threonine­protein kinase hippo and JNK signalling pathways. Together, the results of the present study suggest that PLEKHO1 may contribute to the development of RCC, and therefore, further study is needed to explore its potential as a therapeutic target.


Assuntos
Carcinoma de Células Renais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Renais/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Serina-Treonina Quinases/metabolismo , Adulto , Idoso , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Regulação para Cima
10.
Life Sci ; 231: 116554, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31194992

RESUMO

AIMS: Several adipokines have been proven to improve the therapeutic efficacy of mesenchymal stromal cells (MSCs) when used to treat ischemic heart disease. Asprosin (ASP) is a newly-discovered adipokine. ASP might also predict the severity of coronary pathology. We investigated the role of ASP on MSCs and the effects of ASP-pretreated MSCs on myocardial infarction (MI). MAIN METHODS: MSCs were labelled with a lentivirus carrying green fluorescent protein (GFP). For in vivo study, after pretreatment with vehicle or ASP, MSCs were injected into infarcted hearts. Cardiac function and fibrosis were then evaluated 4 weeks after the induction of MI and survival of MSCs evaluated after 1 week. MSCs proliferation and migration were investigated after ASP treatment in vitro. MSCs apoptosis induced by hydrogen peroxide (H2O2) was assessed using flow cytometry. KEY FINDINGS: Compared to vehicle-pretreated MSCs, ASP-pretreated MSCs significantly improved the left ventricular ejection fraction (LVEF), and inhibited myocardial fibrosis 4 weeks after MI. ASP pretreatment may have promoted homing of transplanted MSCs. In vitro results showed that ASP had no significant effect on MSC proliferation and migration, but protected these cells from H2O2-induced apoptosis. Among 21 molecules associated with antioxidation and cell death, the antioxidant enzyme SOD2 was significantly upregulated by ASP. Furthermore, ASP treatment inhibited H2O2-induced ROS generation and apoptosis via the activated ERK1/2-SOD2 pathway. SIGNIFICANCE: This is the first evidence that ASP can regulate MSCs function and enhance MSCs therapy for ischemic heart disease. Furthermore, we demonstrate that ASP protects MSCs from oxidative stress-induced apoptosis via the ERK1/2-SOD2 pathway.


Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteínas dos Microfilamentos/metabolismo , Infarto do Miocárdio/terapia , Fragmentos de Peptídeos/metabolismo , Hormônios Peptídicos/metabolismo , Superóxido Dismutase/metabolismo , Animais , Apoptose/fisiologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Coração/fisiopatologia , Peróxido de Hidrogênio/farmacologia , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Espécies Reativas de Oxigênio/metabolismo , Função Ventricular Esquerda
11.
Biochemistry (Mosc) ; 84(6): 637-643, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31238863

RESUMO

Taking into account a special role of pancreatic ß-cells in the development of diabetes mellitus, the effects of peroxiredoxin 6 (Prx6) on the viability and functional activity of rat insulinoma RIN-m5F ß-cells were studied under diabetes-simulating conditions. For this purpose, the cells were cultured at elevated glucose concentrations or in the presence of pro-inflammatory cytokines (TNF-α and IL-1) known for their special role in the cytotoxic autoimmune response in diabetes. It was found that the increased glucose concentration of 23-43 mM caused death of 20-60% ß-cells. Prx6 added to cells significantly reduced the level of reactive oxygen species and protected the RIN-m5F ß-cells from hyperglycemia, reducing the death of these cells by several fold. A measurement of insulin secretion by the RIN-m5F ß-cells showed a significant stimulatory effect of Prx6 on the insulin-producing activity of pancreatic ß-cells. It should be noted that the stimulatory activity of Prx6 was detected during culturing the cells under both normal and unfavorable conditions. The regulation of the NF-κB signaling cascade could be one of the mechanisms of Prx6 action on ß-cells, in particular, through activation of RelA/p65 phosphorylation at Ser536.


Assuntos
Citocinas/toxicidade , Glucose/toxicidade , Células Secretoras de Insulina/efeitos dos fármacos , Peroxirredoxina VI/fisiologia , Animais , Morte Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Citocinas/metabolismo , Glucose/metabolismo , Mediadores da Inflamação/metabolismo , Insulina/biossíntese , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Interleucina-1/metabolismo , NF-kappa B/metabolismo , Fosforilação , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
12.
Cornea ; 38(8): 1023-1028, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31090594

RESUMO

PURPOSE: The purpose of this study was to determine the safety of long-term storage and shipping of prestripped, prestained, and preloaded Descemet membrane endothelial keratoplasty (pDMEK) grafts. METHODS: A total of 33 cadaveric corneas were prestripped, prestained, and preloaded using modified Jones tube injectors as pDMEK. The corneas were masked to groups that were prepared <9 hours (control), 48 hours, and 72 hours before unloading and analysis. The 48- and 72-hour tissues were shipped by airfreight on each day before arrival to simulate domestic and international shipping. The corneas were then stained using Calcein AM vital dye (Molecular Probes, Eugene, OR) and imaged using an inverted confocal microscope. Primary outcome measures were endothelial cell loss (ECL, %) and sustainability of staining. MetaMorph software (Molecular Devices, Downingtown, PA) was used to quantify ECL, and staining was evaluated subjectively using all-or-none rating. RESULTS: There was no difference in the mean ECL for the control, 48-hour, and 72-hour groups, which were 25.1% ± 8.8%, 26.4% ± 17.5%, and 19.2% ± 11.5%, respectively (P = 0.45; Kruskal-Wallis test). In all tissues of each group, no loss of staining was identified at each time point of analysis. CONCLUSIONS: ECL in pDMEK tissue prepared 48 and 72 hours in advance and shipped using standard methods is similar to that in pDMEK tissue prepared on the same day. These findings support the safety of domestic and international shipping of pDMEK grafts.


Assuntos
Sobrevivência Celular/fisiologia , Perda de Células Endoteliais da Córnea/fisiopatologia , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior/métodos , Coleta de Tecidos e Órgãos/métodos , Obtenção de Tecidos e Órgãos , Idoso , Contagem de Células , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior/instrumentação , Epitélio Posterior/citologia , Humanos , Microscopia Confocal , Pessoa de Meia-Idade , Preservação de Órgãos/métodos , Doadores de Tecidos , Transportes/métodos
13.
ACS Appl Mater Interfaces ; 11(22): 20437-20452, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31081613

RESUMO

Three-dimensional (3D) printing technology has seen several refinements when introduced in the field of medical devices and regenerative medicines. However, it is still a challenge to 3D print gels for building complex constructs as per the desired shape and size. Here, we present a novel method to 3D print gelatin/carboxymethylchitin/hydroxyapatite composite gel constructs of a complex shape. The objective of this study is to fabricate a bioactive gel scaffold with a controlled hierarchical structure. The hierarchy ranges from 3D outer shape to macroporosity to microporosity and rough surface. The fabrication process developed here uses 3D printing in a local cryogenic atmosphere, followed by lyophilization and cross-linking. The gel instantly freezes after extrusion on the cold plate. The cooling action is not limited to the build plate, but the entire gel scaffold is cooled during the 3D printing process. This enables the construction of a stable self-sustaining large-sized 3D complex geometry. Further, lyophilization introduces bulk microporosity into the scaffolds. The outer shape and macroporosity were controlled with the 3D printer, whereas the microporous structure and desirable rough surface morphology were obtained through lyophilization. With cryogenic 3D printing, up to 90% microporosity could be incorporated into the scaffolds. The microporosity and pore size distribution were controlled by changing the cross-linker and total polymer concentration, which resulted in six times increase in surface open pores of size <20 µm on increasing the cross-linker concentration from 25 to 100 mg/mL. The introduction of bulk microporosity was shown to increase swelling by 1.8 times along with a significant increase in human umbilical cord mesenchymal stem cells and Saos-2 cell attachment (2×), proliferation (2.4×), Saos-2 cell alkaline phosphatase level (2×), and mineralization (3×). The scaffolds are spongy in nature in a wet state, thus making them potential implants for bone cavities with a small opening. The application of these cryogenically 3D printed compressible gel scaffolds with multiscale porosity extends to a small- as well as a large-sized open/partially open patient-specific bone defect.


Assuntos
Osso e Ossos/citologia , Engenharia Tecidual/métodos , Tecidos Suporte/química , Fosfatase Alcalina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Quitina/análogos & derivados , Quitina/química , Gelatina/química , Humanos , Porosidade , Impressão Tridimensional
14.
AAPS PharmSciTech ; 20(5): 198, 2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127389

RESUMO

In this work, several normal, oil-in-water (o/w) microemulsions (MEs) were prepared using peppermint essential oil, jojoba oil, trans-anethole, and vitamin E as oil phases to test their capacity to load paclitaxel (PTX). Initially, pseudo-ternary partial phase diagrams were constructed in order to find the normal microemulsion region using d-α-tocopherol polyethylene glycol 1000 succinate (TPGS-1000) as surfactant and isobutanol (iso-BuOH) as co-surfactant. Selected ME formulations were loaded with PTX reaching concentrations of 0.6 mg mL-1 for the peppermint oil and trans-anethole MEs, while for the vitamin E and jojoba oil MEs, the maximum concentration was 0.3 mg mL-1. The PTX-loaded MEs were stable according to the results of heating-cooling cycles and mechanical force (centrifugation) test. Particularly, drug release profile for the PTX-loaded peppermint oil ME (MEPP) showed that ∼ 90% of drug was released in the first 48 h. Also, MEPP formulation showed 70% and 90% viability reduction on human cervical cancer (HeLa) cells after 24 and 48 h of exposure, respectively. In addition, HeLa cell apoptosis was confirmed by measuring caspase activity and DNA fragmentation. Results showed that the MEPP sample presented a major pro-apoptotic capability by comparing with the unloaded PTX ME sample.


Assuntos
Antineoplásicos Fitogênicos/síntese química , Apoptose/efeitos dos fármacos , Citotoxinas/síntese química , Nanosferas/química , Paclitaxel/síntese química , Óleos Vegetais/síntese química , Antineoplásicos Fitogênicos/farmacocinética , Apoptose/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Citotoxinas/farmacocinética , Relação Dose-Resposta a Droga , Liberação Controlada de Fármacos , Células HeLa , Humanos , Paclitaxel/farmacocinética , Óleos Vegetais/farmacocinética , Polietilenoglicóis/síntese química , Polietilenoglicóis/farmacocinética , Tensoativos/síntese química , Tensoativos/farmacocinética , Vitamina E/síntese química , Vitamina E/farmacocinética
15.
Invest Ophthalmol Vis Sci ; 60(6): 2380-2387, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31141609

RESUMO

Purpose: To determine whether tauopathies are associated with impaired autophagy and involved in the death of retinal ganglion cells (RGCs) of rats from an optic nerve crush (ONC). Methods: Short interfering RNA (siRNA) of the tau gene (si-Tau) or nontargeting siRNA (si-NC) was injected intravitreally 48 hours prior to ONC. The effects of silencing the tau gene on neuroprotection were determined by the number of Tuj-1-stained RGCs on days 7 and 14 after the ONC. The changes in the expressions of phosphorylated tau, P62, and LC3B were determined by immunoblots and immunohistochemistry on day 7. Results: Autophagy was impaired in the retina on day 7 after the ONC as the P62 level increased by 3.1-fold from the sham control level with a reduction in the ratio LC3B2/LC3B1. There was a 2.1-fold increase of phosphorylated tau (ser 396) in the retina, and si-Tau depressed the increase by 1.3-fold (n = 3 each). The expressions of tau and P62 were well colocalized. They were observed in the somas of RGCs and retinal nerve fibers (RNFs), and these expressions were increased after the ONC. Pretreatment by si-Tau showed significant protection in the number of RGCs after the ONC. Specifically, the density of RGCs was 540 ± 74.5 cells/mm2 on day 14 in the si-NC group, while the level was maintained at 1321 ± 192 cells/mm2 in the si-Tau group (n = 4 each). Conclusions: Silencing the tau gene is neuroprotective, and tauopathies may be involved in the death of RGCs after ONC. Impaired autophagy may be involved in ONC-induced tauopathies.


Assuntos
Autofagia/fisiologia , Compressão Nervosa , Neuroproteção/fisiologia , Traumatismos do Nervo Óptico/fisiopatologia , Células Ganglionares da Retina/fisiologia , Proteínas tau/fisiologia , Animais , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Inativação Gênica , Masculino , Ratos , Proteínas tau/metabolismo
16.
Turk Neurosurg ; 29(4): 470-477, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31124572

RESUMO

AIM: To investigate the effect of dabigatran, a new oral anticoagulant, on human primary cell cultures isolated from intact intervertebral disc tissue. MATERIAL AND METHODS: Cell cultures were prepared from tissues obtained from six cases who had undergone surgery due to spinal trauma. Dabigatran, an active pharmacological agent, was applied to intact annulus fibrosus (AF)/nucleus pulposus (NP) primary cell cultures from the study group. After performing cell viability, toxicity, and proliferation tests on all cultures in the control and study groups, the surface morphologies of the samples were evaluated. Subsequently, chondroadherin (CHAD), cartilage oligomeric matrix protein (COMP), and matrix metalloproteinase (MMP)-13 and -19 expressions were measured via a real-time polymerase chain reaction (RT-PCR). Data were analyzed statistically. RESULTS: In the proliferation assays performed on the 20th day of the study, cells in the dabigatran-supplemented group were reported to have lost 46.37% more viability than those in the control group. Expressions of all genes examined except MMP-13 were evaluated in the control group by time, but in contrast to the control group results, COMP and MMP-19 gene expressions decreased in the dabigatran-treated group. No CHAD or MMP-13 expression was noted in these cultures. CONCLUSION: The potential for a systemically applied drug to accumulate in tissue and negatively affect surrounding tissues and microstructures must be emphasized.


Assuntos
Anticoagulantes/efeitos adversos , Dabigatrana/efeitos adversos , Disco Intervertebral/efeitos dos fármacos , Trombose/prevenção & controle , Administração Oral , Adolescente , Adulto , Anticoagulantes/administração & dosagem , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Dabigatrana/administração & dosagem , Proteínas da Matriz Extracelular/metabolismo , Feminino , Expressão Gênica , Humanos , Disco Intervertebral/metabolismo , Disco Intervertebral/cirurgia , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/cirurgia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células/métodos , Trombose/metabolismo , Adulto Jovem
17.
ACS Appl Mater Interfaces ; 11(22): 19712-19723, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31066264

RESUMO

Photonic cancer hyperthermia has been considered to be one of the most representative noninvasive cancer treatments with high therapeutic efficiency and biosafety. However, it still remains a crucial challenge to develop efficient photothermal nanoagents with satisfactory photothermal performance and biocompatibility, among which two-dimensional (2D) ultrathin nanosheets have recently been regarded as the promising multifunctional theranostic agents for photothermal tumor ablation. In this work, we report, for the first time, on the construction of a novel kind of photothermal agents based on the intriguing 2D antimony(III) selenide (Sb2Se3) nanosheets for highly efficient photoacoustic imaging-guided photonic cancer hyperthermia by near-infrared (NIR) laser activation. These Sb2Se3 nanosheets were easily fabricated by a novel but efficiently combined liquid nitrogen pretreatment and freezing-thawing approach, which were featured with high photothermal-conversion capability (extinction coefficient: 33.2 L g-1 cm-1; photothermal-conversion efficiency: 30.78%). The further surface engineering of these Sb2Se3 ultrathin nanosheets with poly(vinyl pyrrolidone) (PVP) substantially improved the biocompatibility of the nanosheets and their stability in physiological environments, guaranteeing the feasibility in photonic antitumor applications. Importantly, 2D Sb2Se3-PVP nanosheets have been certificated to efficiently eradicate the tumors by NIR-triggered photonic tumor hyperthermia. Especially, the biosafety in vitro and in vivo of these Sb2Se3 ultrathin nanosheets has been evaluated and demonstrated. This work meaningfully expands the biomedical applications of 2D bionanoplatforms with a planar topology through probing into new members (Sb2Se3 in this work) of 2D biomaterials with unique intrinsic physiochemical property and biological effect.


Assuntos
Antimônio/química , Substâncias Macromoleculares/química , Nanopartículas/química , Selênio/química , Nanomedicina Teranóstica/métodos , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Humanos , Microscopia Eletrônica de Varredura , Fototerapia/métodos , Polivinil/química , Pirrolidinas/química
18.
BMC Neurosci ; 20(1): 15, 2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30947684

RESUMO

BACKGROUND: Smoking (TS) and recently e-cigarettes (EC) vaping, have been associated with vascular endothelial dysfunction primarily relevant to oxidative stress, exposure to nicotine, and smoking-induced inflammation. It is accepted that both EC and TS enhance glucose intolerance and the risk of developing type-2 diabetes mellitus which is also one of the causes of blood-brain barrier (BBB) damage and the higher risk of cerebrovascular diseases. Recent studies have shown how Metformin, the first common antidiabetic drug, can protect the BBB integrity through enhancement of nuclear factor erythroid 2-related factor (Nrf2) activity. Herein, we investigated the role of rosiglitazone (RSG; family of thiazolidinedione class used oral anti-diabetic drug) in TS/EC-induced BBB impairment. RESULTS: Although the exact mechanism of RSG is not fully understood, previous studies have revealed that RSG can promote counteractive protective mechanisms primarily associated with the enhancement of Nrf2 activity through activation of the peroxisome proliferator-activated receptor gamma. In line with these findings, our results show an increased expression of PPARy by RSG, enhancement of Nrf2 activity and BBB protection against TS/EC exposure including reduced inflammation, oxidative stress, tight junction downregulation and loss of BBB integrity. CONCLUSIONS: RSG could be considered as a promising therapeutic potential to prevent TS/EC induced cerebrovascular dysfunction and possibly other xenobiotic substances which may impact the BBB via oxidative stress-mediated effects. However, additional in vivo studies and clinical setting will be needed to validate our results and assess the full extent of RSG protective effects.


Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Sistemas Eletrônicos de Liberação de Nicotina , Fármacos Neuroprotetores/farmacologia , Rosiglitazona/farmacologia , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Permeabilidade Capilar/fisiologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Camundongos Endogâmicos C57BL , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Microvasos/patologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , PPAR gama/metabolismo , Dados Preliminares , Espécies Reativas de Oxigênio/metabolismo
19.
Life Sci ; 226: 98-106, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980849

RESUMO

AIMS: The acquired drug resistance has been regarded as a main barrier for the effective treatment of temozolomide (TMZ) in glioblastoma (GBM). MiR-126-3p is commonly down-regulated and exerts tumor-suppressive roles in kinds of human cancers, including GBM. This study was designed to investigate the functions and mechanisms of miR-126-3p in regulating TMZ resistance in GBM. MATERIALS AND METHODS: qRT-PCR analysis was used to measure the expressions of miR-126-3p and SOX2 mRNA in GBM tissues and cells. Cell viability, colony forming ability and apoptosis were detected to evaluate the effect of miR-126-3p or SOX2 on TMZ resistance. Luciferase reporter experiments were applied to identify the target genes of miR-126-3p. Western blot analysis was performed to determine the protein levels associated with Wnt/ß-catenin signaling. TOP/FOP Flash assays were conducted to determine the effects of miR-126-3p or SOX2 on Wnt/ß-catenin signaling. KEY FINDINGS: miR-126-3p expression was decreased in TMZ-resistant GBM tissues and cells. High levels of miR-126-3p enhanced TMZ sensitivity by inhibiting cell viability, reducing colony forming potential and inducing apoptosis. Additionally, SOX2 was identified as a downstream target of miR-126-3p. On the contrary, SOX2 overexpression conferred TMZ resistance of GBM cells. Moreover, miR-126-3p-mediated TMZ sensitivity was reversed following increased expression of SOX2. Furthermore, miR-126-3p-induced inactivation of Wnt/ß-catenin signaling was greatly abrogated by SOX2 up-regulation. SIGNIFICANCE: MiR-126-3p sensitizes GBM cells to TMZ possibly by repressing SOX2 expression and blocking Wnt/ß-catenin signaling. This study provides novel targets to overcome TMZ resistance in GBM chemotherapy.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , MicroRNAs/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Temozolomida/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Antineoplásicos Alquilantes/farmacologia , Apoptose/fisiologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , MicroRNAs/biossíntese , MicroRNAs/genética , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/genética
20.
Biomed Pharmacother ; 114: 108825, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30981110

RESUMO

Large tumor suppressor 2 (LATS2), an important mediator of the cell apoptotic response pathway, has been linked to the progression of several cancers. Here, we described the molecular feature of LATS2 as a novel antitumor factor in liver cancer cells in vitro. Western blotting was used to detect the expression of LATS2 and its downstream factors. ELISA, immunofluorescence, and flow cytometry were used to evaluate the alterations of mitochondrial function in response to LATS2 overexpression. Adenovirus-loaded LATS2 and siRNA against DRP1 were transfected into liver cancer cells to overexpress LATS2 and knockdown DRP1 expression, respectively. The results of the present study demonstrated that overexpression of LATS2 was closely associated with more liver cancer cell death. Mechanistically, LATS2 overexpression increased the expression of DRP1, and DRP1 elevated mitochondrial division, an effect that was accompanied by mitochondrial dysfunction, including mitochondrial membrane potential reduction, mitochondrial respiratory complex downregulation, mitochondrial cyt-c release into the nucleus and mitochondrial oxidative injury. Moreover, LATS2 overexpression also initiated mitochondrial apoptosis, and this process was highly dependent on DRP1-related mitochondrial division. Molecular investigations demonstrated that LATS2 modulated DRP1 expression via the Wnt/ß-catenin pathway. Inhibition of the Wnt/ß-catenin pathway pregented LATS2-mediated DRP1 upregulation, ultimately sustaining mitochondrial function and cell viability in the presence of LATS2 overexpression. Altogether, the above data identify LATS2-Wnt/ß-catenin/DRP1/mitochondrial division as a novel anticancer signaling pathway promoting cancer cell death, which might be an attractive therapeutic target for the treatment of hepatocellular carcinoma.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Via de Sinalização Wnt/fisiologia , Apoptose/fisiologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Regulação para Baixo/fisiologia , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia , beta Catenina/metabolismo
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