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1.
Braz J Biol ; 83: e248746, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495165

RESUMO

Colorectal cancer (CRC) is one of the most common cancers leading to comorbidities and mortalities globally. The rational of current study was to evaluate the combined epigallocatechin gallate and quercetin as a potent antitumor agent as commentary agent for therapeutic protocol. The present study investigated the effect of epigallocatechin Gallate (EGCG) (150mg) and quercetin (200mg) at different proportions on proliferation and induction of apoptosis in human colon cancer cells (HCT-116). Cell growth, colonogenic, Annexin V in addition cell cycle were detected in response to phytomolecules. Data obtained showed that, the colony formation was inhibited significantly in CRC starting from the lowest concentration tested of 10 µg/mL resulting in no colonies as visualized by a phase-contrast microscope. Data showed a significant elevation in the annexin V at 100 µg/mL EGCG(25.85%) and 150 µg/mL quercetin (48.35%). Moreover, cell cycle analysis showed that this combination caused cell cycle arrest at the G1 phase at concentration of 100 µg/mL (72.7%) and 150 µg/mL (75.25%). The combined effect of epigallocatechin Gallate and quercetin exert antiproliferative activity against CRC, it is promising in alternative conventional chemotherapeutic agent.


Assuntos
Catequina , Neoplasias Colorretais , Anexina A5 , Catequina/análogos & derivados , Catequina/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Humanos , Quercetina/farmacologia
2.
Cell Physiol Biochem ; 55(4): 449-459, 2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34259420

RESUMO

BACKGROUND/AIMS: Chronic kidney disease is frequently accompanied by anemia, hypoxemia, and hypoxia. It has become clear that the impaired erythropoietin production and altered iron homeostasis are not the sole causes of renal anemia. Eryptosis is a process of red blood cells (RBC) death, like apoptosis of nucleated cells, characterized by Ca2+ influx and phosphatidylserine (PS) exposure to the outer RBC membrane leaflet. Eryptosis can be induced by uremic toxins and occurs before senescence, thus shortening RBC lifespan and aggravating renal anemia. We aimed to assess eryptosis and intracellular oxygen levels of RBC from hemodialysis patients (HD-RBC) and their response to hypoxia, uremia, and uremic toxins uptake inhibition. METHODS: Using flow cytometry, RBC from healthy individuals (CON-RBC) and HD-RBC were subjected to PS (Annexin-V), intracellular Ca2+ (Fluo-3/AM) and intracellular oxygen (Hypoxia Green) measurements, at baseline and after incubation with uremic serum and/or hypoxia (5% O2), with or without ketoprofen. Baseline levels of uremic toxins were quantified in serum and cytosol by high performance liquid chromatography. RESULTS: Here, we show that HD-RBC have less intracellular oxygen and that it is further decreased post-HD. Also, incubation in 5% O2 and uremia triggered eryptosis in vitro by exposing PS. Hypoxia itself increased the PS exposure in HD-RBC and CON-RBC, and the addition of uremic serum aggravated it. Furthermore, inhibition of the organic anion transporter 2 with ketoprofen reverted eryptosis and restored the levels of intracellular oxygen. Cytosolic levels of the uremic toxins pCS and IAA were decreased after dialysis. CONCLUSION: These findings suggest the participation of uremic toxins and hypoxia in the process of eryptosis and intracellular oxygenation.


Assuntos
Eriptose , Eritrócitos/metabolismo , Oxigênio/sangue , Insuficiência Renal Crônica/sangue , Uremia/sangue , Adolescente , Adulto , Idoso , Anexina A5/sangue , Cálcio/sangue , Hipóxia Celular , Eritrócitos/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/patologia , Uremia/patologia
3.
Arch Virol ; 166(9): 2579-2584, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34170427

RESUMO

Airway microparticles (MPs) have been shown previously to inhibit influenza virus by trapping virions on their surface through their surface viral receptor. It was hypothesized that airway MPs may carry most of the epithelial cell surface molecules, including receptors for respiratory viruses, and may be able to inhibit various respiratory viruses. We show here that MPs from human bronchoalveolar lavage (BAL) can inhibit respiratory syncytial virus (RSV). Those MPs stained positive for the RSV receptor, CX3CR1. Furthermore, incubating the MPs with a monoclonal antibody against CX3CR1 reduced the anti-RSV activity. These data indicate that MPs can contribute to respiratory innate antiviral defense.


Assuntos
Antivirais/uso terapêutico , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Sistema Respiratório/virologia , Animais , Anexina A5 , Anticorpos Monoclonais , Anticorpos Antivirais/imunologia , Receptor 1 de Quimiocina CX3C , Micropartículas Derivadas de Células , Células Epiteliais/imunologia , Células Epiteliais/virologia , Humanos , Camundongos , Vírus Sincicial Respiratório Humano/imunologia
4.
Front Immunol ; 12: 647900, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34025656

RESUMO

Acute cellular rejection (ACR) after liver transplantation (LT) goes along with allograft dysfunction, which is diagnosed by liver biopsy and concomitant histological analysis, representing the gold standard in clinical practice. Yet, liver biopsies are invasive, costly, time-intensive and require expert knowledge. Herein we present substantial evidence that blood plasma residing peripheral liver-derived extracellular particles (EP) could be employed to diagnose ACR non-invasively. In vitro experiments showed organ-specific EP release from primary human hepatocytes under immunological stress. Secondly, analysis of consecutive LT patients (n=11) revealed significant heightened EP concentrations days before ACR. By conducting a diagnostic accuracy study (n = 69, DRKS00011631), we explored the viability of using EP as a liquid biopsy for diagnosing ACR following LT. Consequently, novel EP populations in samples were identified using visualization of t-distributed stochastic neighbor embedding (viSNE) and self-organizing maps (FlowSOM) algorithms. As a result, the ASGR1+CD130+Annexin V+ EP subpopulation exhibited the highest accuracy for predicting ACR (area under the curve: 0.80, 95% confidence interval [CI], 0.70-0.90), with diagnostic sensitivity and specificity of 100% (95% CI, 81.67-100.0%) and 68.5% (95% CI, 55.3-79.3%), respectively. In summary, this new EP subpopulation presented the highest diagnostic accuracy for detecting ACR in LT patients.


Assuntos
Anexina A5/sangue , Receptor de Asialoglicoproteína/sangue , Receptor gp130 de Citocina/sangue , Rejeição de Enxerto/sangue , Rejeição de Enxerto/diagnóstico , Transplante de Fígado/efeitos adversos , Adulto , Idoso , Biomarcadores/sangue , Células Cultivadas , Feminino , Hepatócitos/imunologia , Hepatócitos/metabolismo , Humanos , Biópsia Líquida/métodos , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e Especificidade , Transplante Homólogo/efeitos adversos
5.
Methods Mol Biol ; 2318: 209-229, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019292

RESUMO

Although many oncoproteins promote cell growth and proliferation, some also possess the potential to induce cell cycle arrest or cell death by apoptosis. Elevated and deregulated expression of the Myc protein promotes apoptosis in both cultured cells and in some tissues in vivo. Here we describe techniques to detect Myc-induced apoptosis in vitro using flow cytometry, microscopy, and immunoblotting, and in vivo using immunohistochemical staining, immunoblotting, and analysis of RNA expression.


Assuntos
Citometria de Fluxo/métodos , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Anexina A5/metabolismo , Apoptose/genética , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/genética , Morte Celular/genética , Proliferação de Células/genética , DNA/genética , Genes myc , Humanos , Imuno-Histoquímica/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Camundongos , Proteínas Proto-Oncogênicas c-myc/metabolismo
6.
Acta Chim Slov ; 68(1): 151-158, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34057526

RESUMO

Phytoalexins are substances with antimicrobial properties produced by plants after being attacked by microorganisms, especially phytopathogenic fungi and viruses. They are also currently being studied for their antitumor effect. We aimed to study the apoptosis-stimulating effect of homobrassinin and thiazino[6,5-b]indol in human ovarian adenocarcinoma A2780 and A2780cis cells via flow cytometric analysis of annexin V/PI, caspase 3 and 9 activity, cytochrome C release, and smac-diablo accumulation. Using the western blot technique, we also monitored the effect of both indoles on the response of heat shock proteins in these cells. Thiazino[6,5-b]indol showed more pronounced sensitizing and/or pro-apoptotic effect compared to homobrassinin accompanied by increased smac-diablo accumulation at earlier time intervals and pronounced externalization of phosphatidylserine at 72 h in A2780cis compared to A2780 cells. The apoptosis stimulating effect of thiazino[6,5-b]indol in A2780cis cells was associated with significant irreversible downregulation of HSP70 and HSP90 and partly with a decrease of HSP40. On the other hand, cisplatin-induced the apoptosis of sensitive A2780 cells with reversible downregulation of HSP40 and HSP57. In conclusion, the effect of thiazino[6,5-b]indol on resistant A2780cis cells could have a great utility in both the potential prevention and the treatment of other cisplatin-resistant tumor cells.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Indóis/farmacologia , Tiazinas/farmacologia , Tiocarbamatos/farmacologia , Anexina A5/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo
7.
Bioelectrochemistry ; 140: 107837, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34004548

RESUMO

Exposure of living cells to intense nanosecond pulsed electric field (nsPEF) increases membrane permeability to small solutes, presumably by the formation of nanometer-size membrane lesions. Mechanisms responsible for the restoration of membrane integrity over the course of minutes after nsPEF have not been identified. This study explored if ESCRT-III and Annexin V calcium-dependent repair mechanisms, which play critical role in resealing large membrane lesions, are also activated by electroporation and contribute to the membrane resealing. The extent of membrane damage and the time course of resealing were monitored by the time-lapse imaging of propidium (Pr) uptake in human cervical carcinoma (HeLa) cells exposed to trains of 300-ns PEF. The removal of the extracellular Ca2+ slowed down the resealing, although did not prevent it. Recruitment of CHMP4B protein, a component of ESCRT-III complex, to the electroporated plasma membrane was not observed, thus providing no evidence for possible contribution of the macro-vesicle shedding mechanism. In contrast, silencing the AnxA5 gene impaired resealing and reduced the viability of nsPEF-treated cells. We conclude that Annexin V but not ESCRT-III was involved in the repair of HeLa cells permeabilized by 300-ns stimuli, but it was not the only and perhaps not the main repair mechanism.


Assuntos
Anexina A5/metabolismo , Permeabilidade da Membrana Celular , Eletricidade , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Animais , Células CHO , Cricetulus , Células HeLa , Humanos
8.
J Colloid Interface Sci ; 600: 854-864, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34052534

RESUMO

HYPOTHESIS: Annexin A4 and A5 (ANXA4, ANXA5), both shown to be required for efficient plasma membrane repair (PMR) in living cells, bind as trimers to anionic membranes in the presence of calcium. Both annexins induce membrane curvature and self-assemble into crystal arrays on membranes, observations that have been associated with PMR. However, in-vitro studies of annexins have traditionally been performed using single annexins, despite the recruitment of multiple annexins to the damage site in cells. Hence, we study the potential cooperativity of ANXA4 and ANXA5 during membrane binding. EXPERIMENTS: Laser injury experiments were performed on MCF7 cells transfected to transiently express labelled ANXA4 and ANXA5 to study the localization of the proteins at the damage site. Using free-edged DOPC/DOPS (9:1) membranes we investigated the annexin-induced membrane rolling by fluorescence microscopy and the lateral arrangement of annexin trimers on the membrane surface by atomic force microscopy (AFM). FINDING: ANXA4 and ANXA5 colocalise at the damage site of MCF7 cells during repair. A (1:1) mixture of ANXA4 and ANXA5 induces membrane rolling with a time constant intermediate between the value for the pure annexins. While binding of the pure annexins creates crystal lattices, the (1:1) mixture generates a random arrangement of trimers. Thus, curvature induction remains as a functional property of annexin mixtures in PMR rather than crystal formation.


Assuntos
Anexina A4 , Anexinas , Anexina A5 , Anexinas/genética , Cálcio/metabolismo , Membrana Celular/metabolismo
9.
Methods Mol Biol ; 2255: 55-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033094

RESUMO

This chapter describes a real-time, bioluminescent apoptosis assay technique, which circumvents the well-documented "timing condundrum" encountered when employing traditional apoptosis detection chemistries after exposures with inducers of unknown potential. The assay continuously reports the translocation of phosphatidylserine (PS) from the inner membrane leaflet of a cell to the exofacial surface during apoptosis. This homogenous, no-wash, plate-based assay is made possible by two different annexin V fusion proteins, which contain complementing NanoBiT™ luciferase enzyme subunits, a time-released luciferase substrate, and a fluorescent membrane integrity reagent. During apoptosis, luminescence signal is proportional to PS exposure and fluorescence intensity correlated with the degree of secondary necrosis. Altogether, the measures provide exquisite kinetic resolution of dose- and agent-dependent apoptotic responses, from early through late phases. At exposure termination, other compatible reagents can be applied to measure additional orthogonal correlates of cell health.


Assuntos
Anexina A5/metabolismo , Apoptose , Neoplasias da Mama/patologia , Citometria de Fluxo/métodos , Leucemia Mieloide/patologia , Medições Luminescentes/métodos , Neoplasias da Mama/metabolismo , Feminino , Humanos , Leucemia Mieloide/metabolismo , Células Tumorais Cultivadas
10.
BMC Cancer ; 21(1): 438, 2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33879127

RESUMO

BACKGROUND: Daunorubicin is used clinically in the treatment of myeloma, acute lymphatic and myelocytic leukaemia. The toxic lesions caused by daunorubicin induce various modes of cell death, including apoptosis. Apoptosis is highly regulated programmed cell death that can be initiated mainly via two pathways, through death receptors (extrinsic) or involvement of the mitochondria (intrinsic). Induction of apoptosis via these pathways has been alluded following treatment with daunorubicin, but never compared in acute lymphoblastic leukaemia over a time course. METHODS: This study investigated the mechanisms of daunorubicin induced apoptosis in the treatment of CCRF-CEM, MOLT-4 (acute T-lymphoblastic leukaemia) and SUP-B15 (acute B-lymphoblastic leukaemia) cells. Cells were treated with daunorubicin for 4 h, and then placed in recovery medium (without daunorubicin) for 4 h, 12 h and 24 h. Apoptotic response was analysing using annexin-V expression, caspase activity, mitochondrial membrane potential change and an array to detect 43 apoptotic proteins. RESULTS: Daunorubicin induced apoptosis in all leukemic cell lines, but with different levels and duration of response. Both apoptosis levels and caspase activity increased after four hours recovery then declined in CCRF-CEM and MOLT-4 cells. However, SUP-B15 cells displayed initially comparable levels but remained elevated over the 24 h assessment period. Changes in mitochondrial membrane potential occurred in both MOLT-4 and CCRF-CEM cells but not in SUP-B15 cells. Expression of apoptotic proteins, including Bcl-2, Bax, caspase 3 and FADD, indicated that daunorubicin potentially induced both extrinsic and intrinsic apoptosis in both CCRF-CEM and MOLT-4 cells, but only extrinsic apoptosis in SUP-B15 cells. CONCLUSIONS: This study describes variations in sensitivities and timing of apoptotic responses in different leukaemia cell lines. These differences could be attributed to the lack of functional p53 in coordinating the cells response following cytotoxic treatment with daunorubicin, which appears to delay apoptosis and utilises alternative signalling mechanisms that need to be further explored.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Daunorrubicina/farmacologia , Anexina A5/genética , Anexina A5/metabolismo , Antibióticos Antineoplásicos/uso terapêutico , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Daunorrubicina/uso terapêutico , Humanos , Leucemia/tratamento farmacológico , Leucemia/genética , Leucemia/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos
11.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810523

RESUMO

Routine manipulation of the mouse genome has become a landmark in biomedical research. Traits that are only associated with advanced developmental stages can now be investigated within a living organism, and the in vivo analysis of corresponding phenotypes and functions advances the translation into the clinical setting. The annexins, a family of closely related calcium (Ca2+)- and lipid-binding proteins, are found at various intra- and extracellular locations, and interact with a broad range of membrane lipids and proteins. Their impacts on cellular functions has been extensively assessed in vitro, yet annexin-deficient mouse models generally develop normally and do not display obvious phenotypes. Only in recent years, studies examining genetically modified annexin mouse models which were exposed to stress conditions mimicking human disease often revealed striking phenotypes. This review is the first comprehensive overview of annexin-related research using animal models and their exciting future use for relevant issues in biology and experimental medicine.


Assuntos
Anexina A1/metabolismo , Lipídeos/química , Pesquisa Médica Translacional , Animais , Anexina A2/metabolismo , Anexina A5/metabolismo , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Cálcio/química , Membrana Celular/metabolismo , Diabetes Mellitus/metabolismo , Progressão da Doença , Homeostase , Camundongos , Camundongos Knockout , Nanotecnologia , Neoplasias/metabolismo , Neovascularização Patológica , Peptídeos/química , Fenótipo , Ligação Proteica , Transporte Proteico
12.
Nucleic Acids Res ; 49(9): 4864-4876, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33928345

RESUMO

Antisense oligonucleotide (ASO)-based therapy is one of the next-generation therapy, especially targeting neurological disorders. Many cases of ASO-dependent gene expression suppression have been reported. Recently, we developed a tocopherol conjugated DNA/RNA heteroduplex oligonucleotide (Toc-HDO) as a new type of drug. Toc-HDO is more potent, stable, and efficiently taken up by the target tissues compared to the parental ASO. However, the detailed mechanisms of Toc-HDO, including its binding proteins, are unknown. Here, we developed native gel shift assays with fluorescence-labeled nucleic acids samples extracted from mice livers. These assays revealed two Toc-HDO binding proteins, annexin A5 (ANXA5) and carbonic anhydrase 8 (CA8). Later, we identified two more proteins, apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) and flap structure-specific endonuclease 1 (FEN1) by data mining. shRNA knockdown studies demonstrated that all four proteins regulated Toc-HDO activity in Hepa1-6, mouse hepatocellular cells. In vitro binding assays and fluorescence polarization assays with purified recombinant proteins characterized the identified proteins and pull-down assays with cell lysates demonstrated the protein binding to the Toc-HDO and ASO in a biological environment. Taken together, our findings provide a brand new molecular biological insight as well as future directions for HDO-based disease therapy.


Assuntos
Inativação Gênica , Oligonucleotídeos Antissenso/metabolismo , Animais , Anexina A5/metabolismo , Biomarcadores Tumorais/metabolismo , Anidrases Carbônicas/metabolismo , Linhagem Celular , Centrifugação com Gradiente de Concentração , DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases Flap/metabolismo , Polarização de Fluorescência , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Oligonucleotídeos Antissenso/química , RNA , RNA Interferente Pequeno , alfa-Tocoferol
13.
Clin Transl Gastroenterol ; 12(5): e00333, 2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33908373

RESUMO

INTRODUCTION: Microvesicles (MVs) with procoagulant properties may favor liver parenchymal extinction, then cirrhosis-related complications and mortality. In a longitudinal cohort of cirrhotic patients, we measured plasma levels of platelet-derived MVs (PMVs), endothelial-derived MVs, and red blood cell-derived MVs, expressing phosphatidylserine (annexin V-positive [AV+]) or not, and evaluated their impact on Model for End-Stage Liver Disease (MELD) score and transplant-free survival. METHODS: MVs were quantified using flow cytometry in plasma from 90 noninfected cirrhotic patients and 10 healthy volunteers matched for age and sex. Impact of plasma microvesicle levels on 6-month transplant-free survival was assessed using log-rank tests and logistic regression. RESULTS: Microvesicle levels, mostly platelet-derived, were 2.5-fold higher in healthy volunteers compared with cirrhotic patients. Circulating small AV+ PMV levels were lower in cirrhotic patients (P = 0.014) and inversely correlated with MELD scores (R = -0.28; P = 0.0065). During 1-year follow-up, 8 patients died and 7 underwent liver transplantation. In the remaining patients, circulating microvesicle levels did not change significantly. Six-month transplant-free survival was lower in patients with low baseline small AV+ PMV levels (72.6% vs 96.2%; P = 0.0007). In multivariate analyses adjusted for age, ascites, esophageal varices, encephalopathy, clinical decompensation, total platelet counts, MELD score, and/or Child-Pugh C stage, patients with lower small AV+ PMV levels had a significant 5- to 8-fold higher risk of 6-month death or liver transplant. Other PMV levels did not impact on survival. DISCUSSION: Decreased circulating small AV+ PMV levels are associated with significantly lower transplant-free survival in cirrhotic patients independently of MELD score and platelet counts.


Assuntos
Anexina A5/sangue , Plaquetas/metabolismo , Cirrose Hepática/sangue , Microvasos/metabolismo , Idoso , Estudos de Casos e Controles , Endotélio Vascular/metabolismo , Eritrócitos/metabolismo , Feminino , Humanos , Cirrose Hepática/complicações , Cirrose Hepática/cirurgia , Transplante de Fígado , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Análise de Sobrevida
14.
Methods Mol Biol ; 2279: 213-223, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33683697

RESUMO

Annexin V and propidium iodide staining is widely used for determining the cellular death through apoptosis. In the presence of Ca2+ ions, annexin V has a strong binding affinity for phosphatidylserine, a membrane phospholipid that during apoptosis is translocated from the inner side of the cell membrane to its outer side. On the other hand, propidium iodide has ability for DNA binding and it can only enter into necrotic or late apoptotic cells. This chapter describes a commonly used method for detection of apoptosis in a non-small cell lung cancer cell line using annexin V and propidium iodide dye. We describe the detection of different stages of apoptosis in the A549 lung cancer cell line treated with dihydroartemisinin (DHA). This apoptosis detection method can be used to determine the efficacy of different kinds of drugs on cultured cancer cell lines.


Assuntos
Anexina A5 , Apoptose/efeitos dos fármacos , Artemisininas/farmacologia , Carcinoma Pulmonar de Células não Pequenas , Fluoresceína-5-Isotiocianato/análogos & derivados , Neoplasias Pulmonares , Propídio/química , Células A549 , Anexina A5/química , Anexina A5/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia
15.
Mol Med Rep ; 23(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33760105

RESUMO

The present study aimed to determine the anticancer effect of the herbal mixture extract C5E in the pancreatic cancer cell line, PANC­1, in the absence or presence of gemcitabine treatment, a chemotherapeutic drug used for the treatment of pancreatic cancer. The anticancer effects of C5E, gemcitabine and C5E plus gemcitabine in PANC­1 cells following 72 h of treatment were investigated. The effect of each treatment on cell cycle arrest, apoptosis and the proportion of side population (SP) cells was determined using flow cytometric analysis following propidium iodide (PI), Annexin V­FITC/PI double staining and Hoechst 33342 staining, respectively. SP cells share similar characteristics to cancer stem­like cells, and a reduction in the SP is considered to be indicative of an anticancer effect. The percentage of SP cells and the cell viability of general PANC­1 cells were significantly decreased in response to all treatments. The percentage of SP cells was reduced from 8.2% (control) to 3.9, 7.2 and 5.1% following the treatment with C5E, gemcitabine and the co­treatment, respectively. All three treatments were discovered to inhibit cell viability by arresting the cell cycle at the S phase and promoted cell death by inducing early apoptosis, with the levels of apoptosis being increased from 1.9% (control) to 7.3, 2.5 and 12.0% following the treatment with C5E, gemcitabine and the co­treatment, respectively. The mRNA expression levels of sonic hedgehog, which is implicated in the development of certain types of cancer, were downregulated to a greater extent following the co­treatment with C5E and gemcitabine compared with the treatment with either C5E or gemcitabine alone. As the co­treatment with gemcitabine and C5E was more effective than each individual treatment, the present study suggested that the combined treatment may exhibit synergistic effects in PANC­1 cells.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , Extratos Vegetais/farmacologia , Anexina A5/genética , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Fluoresceína-5-Isotiocianato/análogos & derivados , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Medicina Herbária , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Extratos Vegetais/química
16.
J Cell Biochem ; 122(6): 639-652, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33417255

RESUMO

Chrysophanol is an anthraquinone with proven antitumor activity against several tumor cell lines. However, its effect on cervical cancer cells is still unknown. Therefore, HeLa cells were exposed to various concentrations of chrysophanol and then subjected to biochemical, ultrastructural, and morphological analysis. It has been shown using flow cytometry and MTT reduction assay that chrysophanol has been shown to inhibit cell viability and arrest cells in the G2/M phase of the cell cycle. Using Annexin V/propidium iodide staining, a significant increase in apoptosis was found after chrysophanol treatment on HeLa cells, and this process was mediated by caspases 3/7 with a clear inactivation of the antiapoptotic Bcl-2 family protein. However, the demonstrated increased number of cells with double-stranded DNA breaks suggests that chrysophanol also causes DNA damage. By means of electron and fluorescence microscopy, a clear effect of chrysophanol on the intensification of degradation processes, on changes in the structure of the nucleus, endoplasmic reticulum and mitochondria was demonstrated. The changes visible in the mitochondria may be related to the increase in the level of free radicals induced by chrysophanol, which induces apoptosis, inter alia, by increasing the permeability of mitochondrial membranes. The range of observed changes depended on the concentration of anthraquinone was tested.


Assuntos
Antraquinonas/farmacologia , Neoplasias do Colo do Útero/metabolismo , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Dano ao DNA/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo
17.
Phys Chem Chem Phys ; 23(5): 3519-3530, 2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33514968

RESUMO

Glycosaminoglycans (GAGs) are anionic, periodic, linear polysaccharides which are composed of periodic disaccharide units. They play a vital role in many biological processes ongoing in the extracellular matrix. In terms of computational approaches, GAGs are very challenging molecules due to their high flexibility, periodicity, predominantly electrostatic-driven nature of interactions with their protein counterparts and potential multipose binding. Furthermore, the molecular mechanisms underlying GAG-mediated interactions are not fully known yet, and experimental techniques alone are not always sufficient to gain insights into them. The aim of this study was to characterize protein-ion-GAG complexes for the systems where ions are directly involved in GAG binding. Molecular docking, molecular dynamics and free energy calculation approaches were applied to model and rigorously analyse the interactions between annexins (II and V), calcium ions (Ca2+) and heparin (HP). The computational data were examined and discussed in the context of the structural data previously reported by the crystallographic studies. The computational results confirm that the presence of Ca2+ has a tremendous impact on the annexin-HP binding site. This study provides a general computational pipeline to discover the complexity of protein-GAG interactions and helps to understand the role of ions involved at the atomic level. The limitations of the applied protocols are described and discussed pointing at the challenges persisting in the state-of-the-art in silico tools to study protein-ion-GAG systems.


Assuntos
Anexina A2/metabolismo , Anexina A5/metabolismo , Cálcio/metabolismo , Heparina/metabolismo , Animais , Anexina A2/química , Anexina A5/química , Cálcio/química , Heparina/química , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Ratos , Termodinâmica
18.
Methods Mol Biol ; 2240: 243-261, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33423238

RESUMO

Chemical compounds induce cytotoxicity by various mechanisms, including interference in membrane integrity, metabolism, cellular component degradation or release, and cell division. Between the classic death pathways, namely, autophagy, apoptosis, and necrosis, apoptosis have been in the focus for the last several years as an important pathway for the toxicity of different types of xenobiotics. Because of that, having the tools to evaluate it is key for understanding and explaining the toxicodynamics of different classes of substances. Here, we describe a wide array of classic assays that can be easily implemented to evaluate apoptosis induction.


Assuntos
Apoptose/efeitos dos fármacos , Bioensaio , Mitocôndrias/efeitos dos fármacos , Testes de Toxicidade , Animais , Anexina A5/metabolismo , Biomarcadores/metabolismo , Western Blotting , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Fragmentação do DNA , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo
19.
PLoS One ; 16(1): e0245581, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33465114

RESUMO

Evasion of apoptosis is associated with treatment resistance and metastasis in colorectal cancer (CRC). Various cellular processes are associated with evasion of apoptosis. These include overexpression of pro-apoptotic proteins (including p53 and PD-L1), anti-apoptotic proteins (BIRC7/Livin and Bcl-2), chemokine receptors (including DARC), and dysregulation of DNA mismatch repair proteins (including MSH2 and PMS2). The aim of this study was to determine the effect of folinic acid, 5-FU and oxaliplatin (FOLFOX) as a single agent and aspirin plus FOLFOX in various combinations on the aforementioned proteins in human CRC, SW480 cell line and rat models of N-Methyl-N-Nitrosourea (NMU)-induced CRC. In addition, effects of the NMU-induced CRC and chemotherapeutic regimens on haematological and biochemical parameters in the rat models were studied. Immunohistochemistry, immunofluorescence and immunoblot techniques were used to study the expression pattern of the related proteins in the human CRC cells pre- and post-treatment. Double contrast barium enema, post-mortem examination and histological analyses were used to confirm tumour growth and the effect of the treatment in vivo in rat models. Notably, we found in human mucinous CRC, a significant increase in expression of the BIRC7/Livin post-FOLFOX treatment compared with pre-treatment (p = 0.0001). This increase provides new insights into the prognostic role of BIRC7/Livin in evasion of apoptosis and facilitation of treatment resistance, local recurrence and metastasis particularly among mucinous CRCs post-FOLFOX chemotherapy. These poor prognostic features in the CRC may be further compounded by the significant suppression of DARC, PD-L1, PMS2 and overexpression of MSH2 and anti-apoptotic Bcl-2 and p53 proteins observed in our study (p < 0.05). Importantly, we found a significant reduction in expression of BIRC7/Livin and reactivation of DARC and PD-L1 with a surge in Annexin V expression in rat models of CRC cells post-treatment with a sequential dose of aspirin plus FOLFOX compared with other treatments in vivo (p <0.05). The mechanistic rational of these effects underscores the importance of expanded concept of possible aspirin combination therapy with FOLFOX sequentially in future CRC management. Validation of our findings through randomized clinical trials of aspirin plus FOLFOX sequentially in patients with CRC is therefore warranted.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Aspirina/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Anexina A5/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Interações Medicamentosas , Sistema do Grupo Sanguíneo Duffy/metabolismo , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Leucovorina/farmacologia , Leucovorina/uso terapêutico , Masculino , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Neoplasias/metabolismo , Compostos Organoplatínicos/farmacologia , Compostos Organoplatínicos/uso terapêutico , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Receptores de Superfície Celular/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Clin Rheumatol ; 40(7): 2939-2945, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33464429

RESUMO

INTRODUCTION: Besides distinctive respiratory and digestive hallmarks, COVID-19 has been recently associated with a high prevalence of pro-inflammatory and hypercoagulable states known as "COVID-19 Associated Coagulopathy" (CAC), corresponding to a worsening in patients' conditions, whose causes are still to be elucidated. A link between anti-phospholipid antibodies (aPLs) and viral infections has long been suggested. APLs are assessed for anti-phospholipid syndrome (APS) diagnosis, characterized by thrombocytopenia, thrombosis, and coagulopathy. Furthermore, circulating immune complexes (CICs), arisen upon inflammatory responses and related immune dysregulation, can lead to endothelial cell damage and thrombotic complications. METHOD: We performed an extended panel including IgG/IgM anti-cardiolipin, IgG/IgM anti-ß2-glycoprotein-1, coupled with IgG/IgM anti-prothrombin, IgG/IgM anti-annexin-V on two COVID-19 patient groups (early and late infection time), and a negative control group. IgG CIC analysis followed to evaluate inflammatory status, through a possible complement system activation. RESULTS: Our results showed low positive case percentage in IgG/IgM anti-cardiolipin and IgG/IgM anti-ß2-glycoprotein-1 assays (4.54%, 6.25%, and 4.55%; in early infection group, late infection group, and control group, respectively); few positive cases in IgG/IgM anti-prothrombin and IgG/IgM anti-annexin-V immunoassays; and no IgG CIC positivity in any patient. CONCLUSIONS: In conclusion, our data show a low aPL prevalence, likely excluding an involvement in the pathogenesis of CAC. Interestingly, IgG/IgM anti-prothrombin and anti-annexin-V positive cases, detected in late infection group, suggest that aPLs could temporarily increase or could trigger a "COVID-19-induced-APS-like-syndrome" in predisposed patients. Key Points • To our knowledge, anti-prothrombin (aPT) antibodies, anti-annexin-V antibodies and CICs in COVID-19 patients have not been reported in the scientific literature. • Lack of uniformity and the low percentage of aCL/aß2GP1 positivity preclude a putative role in CAC pathogenesis. • IgG/IgM anti-prothrombin and IgG/IgM anti-annexin-V data show that distribution of positive case number increases in late infection patients, significantly in anti-annexin-V results, suggesting a possible role for these anti-phospholipid antibodies in disease course. • aPLs can arise transiently in some patients with critical illness and SARS-CoV-2 infection (disappearing in a few weeks), as well as in other genetically predisposed patients; they could trigger a "COVID-19-induced-APS-like-syndrome".


Assuntos
Síndrome Antifosfolipídica , COVID-19 , Anexina A5 , Anticorpos Anticardiolipina , Complexo Antígeno-Anticorpo , Humanos , SARS-CoV-2
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