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1.
Zhongguo Zhen Jiu ; 40(7): 749-55, 2020 Jul 12.
Artigo em Chinês | MEDLINE | ID: mdl-32648400

RESUMO

OBJECTIVE: To observe the impacts of electroacupuncture (EA) on neurological function, the pathological morphology in brain tissue, apoptosis level and the protein expressions of apoptosis-related cytochrome C (Cyt-C) and cysteine aspartic acid protease-9 (Caspase-9) in the rats with traumatic brain injury (TBI) and explore the potential mechanism of EA in treatment of TBI. METHODS: A total of 70 clean-grade SD mice were randomized into a blank group (8 rats), a sham-operation group (8 rats), a model group (27 rats) and an EA group (27 rats). In terms of interventions of 3, 7 and 14 days, 3 subgroups were divided in the model group and the EA group successively, 9 rats in each subgroup. The modified Feeney free-fall percussion method was adopted to establish TBI models of rats. In the sham-operation group, only the skull was exposed and drilled and no free-fall percussion was exerted. One day after modeling, EA was given in the rats of EA group at "Shuigou" (GV 26), "Baihui" (GV 20) and "Neiguan" (PC 6) and "Zusanli" (ST 36) on the affected side, with intermittent wave, 2 Hz in frequency, once daily, 10 min each time, for 3, 7 and 14 days successively. Separately, on the day 3, 7 and 14 of intervention, the modified neurological severity scale (mNSS) was used to evaluate the degree of neurological function injury in the rats, HE staining and Nissl staining were to observe the pathological and morphological changes in brain tissue, TUNEL method was to observe the level of apoptosis in brain tissue and immunohistochemistry (IHC) method and Western blot were to determine the protein expressions of Cyt-C and Caspase-9 in brain tissue. RESULTS: Compared with the sham-operation group, on the day 3, 7 and 14 of intervention, mNSS scores were increased obviously in the rats of the model group respectively (P<0.01). Compared with the model group, on the day 3, 7 and 14 of intervention, mNSS scores were reduced in the rats of the EA group respectively (P<0.05). On day 3 of intervention, in brain injury region of the rats in the model group and the EA group, gross tissue necrosis, nuclear fragmentation, consolidation and obvious vacuolar changes, reduced Nissl bodies and scattered arrangement were found. On day 7 and 14 of intervention, in the model group and the EA group, the new connective tissue filling and normal cells were visible and Nissl bodies increased. The overall repair and Nissl body quantity in the EA group were better than the model group. Compared with the sham-operation group, on day 3, 7 and 14 of intervention, the numbers of apoptotic cells were increased obviously in the model group (P<0.01) and they were reduced in the EA group as compared with the model group (P<0.05). Compared with the sham-operation group, on day 3, 7 and 14 of intervention, the protein expressions of Cyt-C and Caspase-9 in damaged brain tissue were all increased obviously in the model group (P<0.01) and they were all reduced in the EA group as compared with the model group successively (P<0.05). CONCLUSION: Electroacupuncture remarkably improves the condition in the neurological function injury and reduces apoptosis degree in TBI model rats, which is likely related to the down-regulation of the protein expressions of Cyt-C and Caspase-9 in damaged brain tissue and further to bring the impacts on mitochondria mediated apoptosis process.


Assuntos
Apoptose , Lesões Encefálicas Traumáticas/terapia , Eletroacupuntura , Animais , Caspase 9/metabolismo , Citocromos c/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley
2.
Nat Commun ; 11(1): 3173, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576823

RESUMO

Central nervous system ischemic injury features neuronal dysfunction, inflammation and breakdown of vascular integrity. Here we show that activation of endothelial caspase-9 after hypoxia-ischemia is a critical event in subsequent dysfunction of the blood-retina barrier, using a panel of interrelated ophthalmic in vivo imaging measures in a mouse model of retinal vein occlusion (RVO). Rapid nonapoptotic activation of caspase-9 and its downstream effector caspase-7 in endothelial cells promotes capillary ischemia and retinal neurodegeneration. Topical eye-drop delivery of a highly selective caspase-9 inhibitor provides morphological and functional retinal protection. Inducible endothelial-specific caspase-9 deletion phenocopies this protection, with attenuated retinal edema, reduced inflammation and preserved neuroretinal morphology and function following RVO. These results reveal a non-apoptotic function of endothelial caspase-9 which regulates blood-retina barrier integrity and neuronal survival, and identify caspase-9 as a therapeutic target in neurovascular disease.


Assuntos
Caspase 9/metabolismo , Hipóxia/metabolismo , Isquemia/metabolismo , Oclusão da Veia Retiniana/metabolismo , Lesões do Sistema Vascular/metabolismo , Animais , Barreira Hematorretiniana/metabolismo , Caspase 7/metabolismo , Caspase 9/efeitos dos fármacos , Caspase 9/genética , Morte Celular , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Feminino , Predisposição Genética para Doença/genética , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coelhos , Retina/metabolismo , Retina/patologia , Oclusão da Veia Retiniana/tratamento farmacológico , Oclusão da Veia Retiniana/patologia , Lesões do Sistema Vascular/patologia
3.
Cell Immunol ; 354: 104145, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32569876

RESUMO

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen known to persist in host cells. The apoptotic response of macrophages serves as a defense mechanism to inhibit the growth of intracellular bacteria, the failure of which can favor the spread of the pathogen to new cells. However, the mycobacterial components that regulate cell death and the related underlying mechanisms remain poorly understood. In this study, we investigated protein Rv3261, isolated from an Mtb culture filtrate, for its apoptotic potential using multidimensional fractionation. Rv3261 was found to induce macrophage apoptosis through the caspase-3/-9-dependent pathway. Furthermore, the ROS-dependent JNK activation pathway was found to be critical in Rv3261-mediated apoptosis. Rv3261 inhibited the growth of intracellular Mtb, which was significantly abrogated by pre-treatment with the ROS scavenger N-acetylcysteine (NAC), suggesting that Rv3261-mediated apoptosis may act as a host defense response. These findings suggest that Rv3261 is involved in the apoptotic modulation of Mtb-infected macrophages.


Assuntos
Proteínas de Bactérias/metabolismo , Macrófagos/microbiologia , Mitocôndrias/metabolismo , Mycobacterium tuberculosis/fisiologia , Acetilcisteína/farmacologia , Animais , Apoptose , Caspase 3/metabolismo , Caspase 9/metabolismo , Processos de Crescimento Celular , Evasão da Resposta Imune , Imunidade Inata , Espaço Intracelular , MAP Quinase Quinase 4/metabolismo , Macrófagos/imunologia , Camundongos , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
4.
Medicine (Baltimore) ; 99(17): e19848, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32332640

RESUMO

Xiakemycin A (XKA), a new antibiotic in the pyranonaphthoquinone family, shows antitumor activity. However, the type of cell death induced by XKA remains elusive. In this study, we aim to investigate the type of death induced by XKA in hepatic cancer.The apoptotic features, such as chromatic agglutination, reactive oxygen species generation and membrane potential of mitochondria, in HepG2 cells treated by XKA were measured by Hoechst 33342 staining and flow cytometry. Apoptosis of HepG2 cells treated with XKA was determined by Annexin V-FITC/propidium iodide double staining and Western blot analysis, respectively.XKA had a significant dose-dependent elevation of chromatic agglutination, reactive oxygen species generation, Annexin V and propidium iodide staining, decrease of membrane potential. Meanwhile, in apoptotic HepG2 cells induced by XKA, robust increment was noticed in p53 expression, cleavage of PARP, caspase-3, and caspase-9.XKA showed potent inhibitory effects on the proliferation of HepG2 cells. Such phenomenon may be related to activation of the apoptotic pathway.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Naftoquinonas/farmacologia , Anexina A5/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Caspase 3/metabolismo , Caspase 9/metabolismo , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/fisiologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Propídio/metabolismo , Espécies Reativas de Oxigênio/metabolismo
5.
Chem Biol Interact ; 323: 109075, 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32229109

RESUMO

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/metabolismo
6.
PLoS One ; 15(4): e0223208, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302311

RESUMO

The aim of this study was to investigate whether exogenous erythropoietin (EPO) administration attenuates N-methyl-D-aspartate (NMDA)-mediated excitotoxic retinal damage in Wistar rats. The survival rate of retinal ganglion cells (RGCs) were investigated by flat mount analysis and flow cytometry. A total of 125 male Wistar rats were randomly assigned to five groups: negative control, NMDA80 (i.e., 80 nmoles NMDA intravitreally injected), NMDA80 + 10ng EPO, NMDA80 + 50ng EPO, and NMDA80 + 250ng EPO. The NMDA80 + 50ng EPO treatment group was used to evaluate various administrated points (pre-/co-/post- administration of NMDA80). Meanwhile, the transferase dUTP Nick-End Labeling (TUNEL) assay of RGCs, the inner plexiform layer (IPL) thickness and the apoptotic signal transduction pathways of µ-calpain, Bax, and caspase 9 were assessed simultaneously using an immunohistochemical method (IHC). When EPO was co-administered with NMDA80, attenuated cell death occurred through the downregulation of the apoptotic indicators: µ-calpain was activated first (peak at ~18hrs), followed by Bax and caspase 9 (peak at ~40hrs). Furthermore, the images of retinal cross sections have clearly demonstrated that thickness of the inner plexiform layer (IPL) was significantly recovered at 40 hours after receiving intravitreal injection with NMDA80 and 50ng EPO. Exogenous EPO may protect RGCs and bipolar cell axon terminals in IPL by downregulating apoptotic factors to attenuate NMDA-mediated excitotoxic retinal damage.


Assuntos
Apoptose , Eritropoetina/farmacologia , N-Metilaspartato/farmacologia , Fármacos Neuroprotetores/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Caspase 9/genética , Caspase 9/metabolismo , Regulação para Baixo , Masculino , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/metabolismo , Células Ganglionares da Retina/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
7.
Zhongguo Zhong Yao Za Zhi ; 45(6): 1418-1422, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32281356

RESUMO

Polyphyllin D is a steroid saponin monomer in Polyphyllin, with antibacterial, analgesic, sedative, anti-tumor and other pharmacological effects, but is rarely reported in pancreatic cancer. This study detected apoptosis-relevant indicators, in order to explore the effect of polyphyllin D on the proliferation and apoptosis of human pancreatic cancer Panc-1 cells and relevant mechanisms of action. After pancreatic cancer Panc-1 cells were treated with polyphyllin D(0, 1, 2, 3, 4, 5 µg·µL~(-1)) for 24, 48 and 72 hours, CCK-8 method was used to detect the effect of polyphyllin D on the proliferation of pancreatic cancer Panc-1 cells. Flow cytometry was used to detect cell cycle and changes in mitochondrial membrane potential(MMP). The apoptosis was detected by Annexin V-FITC/PI staining, and Western blot was used to detect the protein expressions of cytochrome C(Cyto C), Bax, Bcl-2, cleaved caspase-3 and cleaved caspase-9. The results indicated that compared with the control group, polyphyllin D could inhibit the proliferative activity of Panc-1 cells in a time and concentration-dependent manner. Flow cytometry results showed that polyphyllin D could block the cells in S and G_2/M phase in a concentration manner, the MMP of the cells was significantly reduced, and the apoptosis rate increased with the concentration of polyphyllin D. Western blot results showed that polyphyllin D could concentration-dependently up-regulate the protein expression levels of Bax, Cyto C, cleaved caspase-3 and cleaved caspase-9, and down-regulate the protein expression level of Bcl-2. The above findings suggested that polyphyllin D could effectively inhibit the proliferation of Panc-1 cells, and its mechanism may be related to the blocking of cell growth cycle and the apoptosis induced by mitochondrial pathway.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose , Diosgenina/análogos & derivados , Neoplasias Pancreáticas/patologia , Saponinas/farmacologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Diosgenina/farmacologia , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
8.
Nat Immunol ; 21(5): 546-554, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32231300

RESUMO

High-dose radiation activates caspases in tumor cells to produce abundant DNA fragments for DNA sensing in antigen-presenting cells, but the intrinsic DNA sensing in tumor cells after radiation is rather limited. Here we demonstrate that irradiated tumor cells hijack caspase 9 signaling to suppress intrinsic DNA sensing. Instead of apoptotic genomic DNA, tumor-derived mitochondrial DNA triggers intrinsic DNA sensing. Specifically, loss of mitochondrial DNA sensing in Casp9-/- tumors abolishes the enhanced therapeutic effect of radiation. We demonstrated that combining emricasan, a pan-caspase inhibitor, with radiation generates synergistic therapeutic effects. Moreover, loss of CASP9 signaling in tumor cells led to adaptive resistance by upregulating programmed death-ligand 1 (PD-L1) and resulted in tumor relapse. Additional anti-PD-L1 blockade can further overcome this acquired immune resistance. Therefore, combining radiation with a caspase inhibitor and anti-PD-L1 can effectively control tumors by sequentially blocking both intrinsic and extrinsic inhibitory signaling.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Caspase 9/metabolismo , Inibidores de Caspase/uso terapêutico , Quimiorradioterapia/métodos , Neoplasias Colorretais/terapia , Ácidos Pentanoicos/uso terapêutico , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Caspase 9/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transplante de Neoplasias , Transdução de Sinais , Regulação para Cima
9.
Cell Physiol Biochem ; 54(3): 354-370, 2020 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-32298553

RESUMO

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - RNA-guided Cas9 endonuclease system has provided a fast and efficient method for precise genome editing in diverse mammalian species, including humans. The CRISPR/Cas9 technology allows generation of modifications into site-specific locations of the selected genes in one major step by carrying deletions, insertions or DNA donor-directed precise sequence modifications. Cas9 forms a nucleoprotein complex with a sequence-specific guide RNA to create double-stranded breaks in complementary DNA target. Further, double-stranded break repair machinery leads to the intended gene modifications. The CRISPR/Cas9 system is widely used technique for genome modification, editing and other biotechnology applications, such as functional annotation, a system for visualization of specific genomic loci and transcriptional control of genes. CRISPR/Cas9-mediated manipulation of the laboratory animal genomes has contributed to the understanding of gene functions and has become a popular approach for modeling human disorders. Furthermore, the growing application of CRISPR-Cas9 system to human genes emerges as an extremely powerful technology for the molecular characterization and treatment of human disease. In this review we present the essential principles of CRISPR/Cas9 technology and the recent advances in its use in translational biomedicine.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Regulação da Expressão Gênica/genética , Terapia Genética/métodos , Animais , Sistemas CRISPR-Cas/fisiologia , Caspase 9/química , Caspase 9/genética , Caspase 9/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Modelos Animais de Doenças , Engenharia Genética/métodos , Recombinação Homóloga/genética , Humanos , RNA Guia/genética
10.
Arch Biochem Biophys ; 683: 108324, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32112740

RESUMO

Glaucoma is the leading cause of irreversible blindness in the world and trabeculectomy remains still the most commonly performed filtration surgery. Failure of trabeculectomy is due to the formation of scarring, which is associated with the increased fibroblast proliferation, activation, and collagen deposition at the site of the drainage channel with subconjunctival fibrosis. Our previous study has revealed that zinc oxide (ZnO) nanoparticles could efficiently decrease the expressions of TGF-ß1 and inhibit fibroblast-mediated collagen lattice contraction. However, the mechanism underlying ZnO nanoparticle-induced fibroblast apoptosis is still unclear. In the present study, we investigated the effect of ZnO nanoparticles on the reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) in human Tenon fibroblasts (HTFs). Moreover, we also explored the influence of ZnO nanoparticles on the expression of Caspase-3, Caspase-9, apoptotic protease-activating factor-1 (Apaf-1), fibroblast-specific protein-1 (FSP-1), collagen III, and E-cadherin. The results indicated that ZnO nanoparticles markedly inhibit HTFs viability and decrease the Δψm in a concentration-dependent pattern. Exposure of HTFs to ZnO nanoparticles could also induce the elevated Caspase-3, Caspase-9, and Apaf-1 expression, decrease the levels of FSP-1, collagen III, and E-cadherin expression, leading to HTFs apoptosis. Our results suggested that elevated ROS and activated Caspase signaling play a fundamental role in ZnO nanoparticle-induced HTFs apoptosis.


Assuntos
Apoptose , Fibroblastos/citologia , Nanopartículas Metálicas/química , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Óxido de Zinco/química , Antioxidantes/metabolismo , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Movimento Celular , Sobrevivência Celular , Humanos , Potencial da Membrana Mitocondrial , Fator de Crescimento Transformador beta1/metabolismo
11.
Arch Biochem Biophys ; 684: 108327, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32142890

RESUMO

Endometrial carcinoma is a type of gynecological cancer that originates in the endometrial epithelial tissue. Due to its high proliferation and ability to invade muscle tissue, it is one of the most common malignant tumors in the female reproductive system. Fatostatin is a small molecule non-sterol diarylthiazole derivative that acts as a chemical inhibitor of the sterol regulatory-element binding protein (SREBP) pathway. Previous studies have shown that fatostatin has an anti-tumor effect in some cancers. In this study, we investigated the effect of fatostatin on the growth, proliferation, apoptosis, migration and cell cycle of human endometrial carcinoma cells (HEC-1A and AN3 CA cells) using cholecystokinin (CCK) -8 method, clonogenicity assay, wound closure assay, Transwell migration assay and flow cytometer. We also examined its effect on the expression of apoptosis-associated protein (Caspase-3, Caspase-8 and Caspase-9) and level of lipid metabolism-related proteins, free fatty acid, and total cholesterol in cells. The growth of endometrial carcinoma xenografts was measured to confirm the effect of fatostatin in vivo. Our results showed that fatostatin inhibited the growth and proliferation of human endometrial carcinoma cells, changed their cell cycle and induced apoptosis. Based on the preliminary animal experiments, fatostatin also exhibited antitumor activity. The present study adds a new dimension to our understanding of the antitumor effects of fatostatin and provides an experimental basis for its use, and supports its potential value for clinical application.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Endométrio/tratamento farmacológico , Metabolismo dos Lipídeos/efeitos dos fármacos , Piridinas/uso terapêutico , Tiazóis/uso terapêutico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Feminino , Humanos , Camundongos Endogâmicos BALB C , Piridinas/farmacologia , Tiazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Arch Med Res ; 51(3): 224-232, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32147288

RESUMO

BACKGROUND: Investigation into the anti-cancer activities of natural products and their derivatives represents an efficient approach to develop safe and effective chemotherapeutic agents for the treatment of colorectal cancer. Helveticoside is a biologically active component of the seed extract of Descurainia sophia. This compound has been reported to regulate the genes related to cell proliferation and apoptosis in lung cancer cells, however its anticancer activity has not been fully explored yet. METHODS: Cell viability was evaluated by MTT and Trypan blue exclusion assay; cell apoptosis was measured by flow cytometry; mitochondrial membrane potential was determined by using JC1-mitochondrial membrane potential assay kit; protein levels were determined by western blot assay; in vivo tumor growth was assessed in a xenograft nude mice model. RESULTS: The current study demonstrated the in vitro anti-cancer activity of helveticoside against colorectal cancer using colorectal cancer cells SW480 and HCT116. Moreover, induction of apoptosis was found to mediate the cytotoxic action of helveticoside on SW480 and HCT116 cells. Based on the decrease in the mitochondrial membrane potential, upregulation of Bax, downregulation of Bcl-2 and cleavage of caspase-3 and 9, apoptosis was induced by helveticoside via mitochondria-mediated intrinsic apoptotic signaling pathways in colorectal cancer cells. Besides, using p53-knockout SW480 cells, the cytotoxic action of helveticoside was found to be p53-dependent. More importantly, administration of helveticoside inhibited the growth of HCT116 cells derived-colorectal cancer xenograft in mice via activation of apoptosis. CONCLUSIONS: Helveticoside might be a potential candidate for the development of novel chemotherapeutic agents for the treatment of colorectal cancer, while the potential toxic effects of helveticoside may be worthy of further investigations.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Glicosídeos Digitálicos/farmacologia , Estrofantinas/farmacologia , Animais , Antineoplásicos/efeitos adversos , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Glicosídeos Digitálicos/efeitos adversos , Células HCT116 , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Nus , Camundongos SCID , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Estrofantinas/efeitos adversos , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/metabolismo
13.
Clin Interv Aging ; 15: 373-385, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32214804

RESUMO

Objective: To elucidate the expression and function of miR-34a in rat osteoarthritic cartilage cells, and further to explore its mechanism. Material and Methods: Rat model of osteoarthritis was constructed and knee joint cartilage cells were isolated in vitro. Immunocytochemical staining was used for identification. qRT-PCR was used to detect the expression of miR-34a in cartilaginous tissues and cartilage cells. Cartilage cells were divided into blank control (BC), negative control (NC), miR-34a inhibitor (34aI), osteoarthritis model (OA), osteoarthritis model + negative control (OA + NC) and osteoarthritis model + miR-34a inhibitor (OA + 34aI) groups. Cell proliferation was detected by CCK-8 and colony formation assays. Cell apoptosis was studied by flow cytometry and Western blot. PI3K/AKT-pathway-related proteins were also analyzed by Western blot. To further validate the effect of miR-34a on the PI3K/Akt pathway, the cartilage cells were divided into blank control (BC), osteoarthritis model (OA), osteoarthritis model + miR-34a inhibitor (OA + 34aI), osteoarthritis model + PI3K activator (OA + IGF-1) and osteoarthritis model + miR-34a inhibitor + PI3K inhibitor (OA + 34aI + LY) groups, the experiments above were repeated. Results: The expression of miR-34a in cartilaginous tissues and cells of osteoarthritis model was significantly higher than that in normal (p < 0.05). After silencing miR-34a gene, the cell proliferation and proteins expression of PI3K/Akt pathway were increased, while the apoptosis rate and expression of apoptosis-related proteins were decreased. Addition of PI3K activator also evidently promoted proliferation and inhibited apoptosis. The protein expression of Bax, Cleaved caspase-3 and Cleaved caspase-9 were dramatically decreased, while the ratios of p-PI3K/PI3K and p-Akt/Akt were increased in OA + IGF-1 group. Conclusion: Downregulation of miR-34a regulated proliferation and apoptosis of cartilage cells by activating PI3K/Akt pathway, providing a potential therapeutic approach for the treatment of osteoarthritis.


Assuntos
Condrócitos/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoartrite do Joelho/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Proliferação de Células , Condrócitos/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Inativação Gênica , Fator de Crescimento Insulin-Like I/farmacologia , Masculino , MicroRNAs/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Ratos , Transdução de Sinais , Proteína X Associada a bcl-2/metabolismo
14.
Biochem Pharmacol ; 176: 113902, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32156660

RESUMO

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the fourth most frequent cause of cancer-related death worldwide. Sorafenib is the first line recommended therapy for patients with locally advanced/metastatic HCC. The low response rate is attributed to intrinsic resistance of HCC cells to Sorafenib. The potential resistance to Sorafenib-induced cell death is multifactorial and involves all hallmarks of cancer. However, the presence of sub-therapeutic dose can negatively influence the antitumoral properties of the drug. In this sense, the present study showed that the sub-optimal Sorafenib concentration (10 nM) was associated with activation of caspase-9, AMP-activated protein kinase (AMPK), sustained autophagy, peroxisome proliferator-activated receptor-coactivator 1α (PGC-1α) and mitochondrial function in HepG2 cells. The increased mitochondrial respiration by Sorafenib (10 nM) was also observed in permeabilized HepG2 cells, but not in isolated rat mitochondria, which suggests the involvement of an upstream component in this regulatory mechanism. The basal glycolysis was dose dependently increased at early time point studied (6 h). Interestingly, Sorafenib increased nitric oxide (NO) generation that played an inhibitory role in mitochondrial respiration in sub-therapeutic dose of Sorafenib. The administration of sustained therapeutic dose of Sorafenib (10 µM, 24 h) induced mitochondrial dysfunction and dropped basal glycolysis derived acidification, as well as increased oxidative stress and apoptosis in HepG2. In conclusion, the accurate control of the administered dose of Sorafenib is relevant for the potential prosurvival or proapoptotic properties induced by the drug in liver cancer cells.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sorafenibe/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Caspase 9/metabolismo , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Masculino , Mitocôndrias Hepáticas/metabolismo , Óxido Nítrico/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ratos Wistar
15.
Oxid Med Cell Longev ; 2020: 7639109, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32190177

RESUMO

This study assessed the protective mechanism of astaxanthin (ASX) against ochratoxin A- (OTA-) induced cardiac injury in mice. Four groups of mice were established: control group (0.1 mL olive oil + 0.1 mL NaHCO2), OTA group (0.1 mL OTA 5 mg/kg body weight), ASX group (0.1 mL ASX 100 mg/kg body weight), and ASX + OTA group (0.1 mL ASX 100 mg/kg body weight, 2 h later, 0.1 mL OTA 5 mg/kg body weight). The test period lasted for 27 days (7 days of dosing, 2 days of rest). Electrocardiogram, body weight, heart weight, tissue pathology, oxidative markers (malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH)), biochemical markers (creatine kinase (CK), creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase (LDH)), electron microscopy, TUNEL, and Western blot tests were used to examine the effects of OTA on myocardial injury and ASX detoxification. The results showed that OTA exposure significantly decreased both body weight and heart weight. OTA induced a decrease in heart rate in mice and decreased tissue concentrations of SOD, CAT, and GSH, while increasing serum concentrations of cardiac enzymes (CK, CK-MB, and LDH) and tissue MDA. ASX improved heart rate, cardiac enzymes, and antioxidant levels in mice. The results of tissue pathology and TUNEL assay showed that ASX protects against OTA-induced myocardial injury. In addition, Western blot results showed that the OTA group upregulated Keap1, Bax, Caspase3, and Caspase9, while it downregulated Nrf2, HO-1, and Bcl-2 protein expression. ASX played a protective role by changing the expression of Keap1, Nrf2, HO-1, Bax, Bcl-2, Caspase3, and Caspase9 proteins. These results indicate that the protective mechanism of ASX on the myocardium works through the Keap1-Nrf2 signaling pathway and mitochondria-mediated apoptosis pathway. This study provides a molecular rationale for the mechanism underlying OTA-induced myocardial injury and the protective effect of ASX on the myocardium.


Assuntos
Apoptose/efeitos dos fármacos , Cardiotônicos/farmacologia , Miocárdio/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Transdução de Sinais , Animais , Antioxidantes/metabolismo , Peso Corporal/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 9/metabolismo , Eletrocardiografia , Frequência Cardíaca/efeitos dos fármacos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Miocárdio/ultraestrutura , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ocratoxinas , Tamanho do Órgão/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Xantofilas/farmacologia , Proteína X Associada a bcl-2/metabolismo
16.
J Exp Clin Cancer Res ; 39(1): 32, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-32039741

RESUMO

BACKGROUND: Exosomes are essential for tumor growth, metastasis, and are used as novel signaling molecules in targeted therapies. Therefore, exosomal miRNAs can be used in new diagnostic and therapeutic approaches due to their involvement in the development of cancers. However, the detailed biological function, potential molecular mechanism and clinical application of exo-miR-15b-3p in gastric cancer (GC) remains unclear. METHODS: miR-15b-3p mRNA levels in tissues, serum, cells and exosomes were analyzed using qRT-PCR assays. qRT-PCR, immunohistochemical and western blotting analyses were utilized for the determination of DYNLT1 expression. The interrelationship connecting miR-15b-3p with DYNLT1 was verified using Dual-luciferase report, western blotting and qRT-PCR assays. Fluorescent PKH-26 or GFP-Lv-CD63 labeled exosomes, as well as Cy3-miR-15b-3p, were utilized to determine the efficacy of the transfer of exo-miR-15b-3p between BGC-823 and recipient cells. Several in vitro assays and xenograft tumor models were conducted to determine exo-miR-15b-3p impact on GC progression. RESULTS: This is the first study to confirm high miR-15b-3p expression in GC cell lines, tissues and serum. Exosomes obtained from 108 GC patient serum samples and GC cell-conditioned medium were found to show upregulation of exo-miR-15b-3p, with the area under the ROC curve (AUC) being 0.820 [0.763-0.876], which is superior to the AUC of tissues and serum miR-15b-3p (0.674 [0.600-0.748] and 0.642 [0.499-0.786], respectively). In addition, high exo-miR-15b-3p expression in serum was found to accurately predict worse overall survival. SGC-7901 and GES-1 cells are capable of internalizing BGC-823 cell-derived exosomes, allowing the transfer of miR-15b-3p. Migration, invasion, proliferation and inhibition of apoptosis in vitro and in vivo were enhanced by exo-miR-15b-3p, by restraining DYNLT1, Cleaved Caspase-9 and Caspase-3 expression. CONCLUSIONS: This study identified a previously unknown regulatory pathway, exo-miR-15b-3p/DYNLT1/Caspase-3/Caspase-9, which promotes GC development and GES-1 cell malignant transformation. Therefore, serum exo-miR-15b-3p may be a potential GC diagnosis and prognosis biomarker, which can be used in precise targeted GC therapy.


Assuntos
Caspase 3/metabolismo , Caspase 9/metabolismo , Transformação Celular Neoplásica/genética , Dineínas/metabolismo , Exossomos/metabolismo , MicroRNAs/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Idoso , Animais , Apoptose/genética , Biomarcadores Tumorais , Caspase 9/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Masculino , Camundongos , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Transporte de RNA , Transdução de Sinais , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/mortalidade
17.
Biomed Pharmacother ; 122: 109712, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31918281

RESUMO

BACKGROUND: Human multiple myeloma (MM) is a malignant and incurable B cell tumor. Zinc oxide nanoparticles (ZnO NPs) have been widely used in biomedical fields including anti-bacterial and anti-tumor. However, the influence of ZnO NPs on MM cells is still unclear. The present study aimed to investigate the effect of ZnO NPs on MM cell (a human myeloma-derived RPMI8226 cell line) death in vitro and the underlying mechanism. METHODS: The morphology of ZnO NPs was characterized by transmission electron microscopy (TEM), and the inhibitory and apoptotic effect of ZnO NPs on human MM cells was monitored by a CCK-8 method and an Annexin V-FITC/PI assay. Meanwhile, the morphological change in the cells after exposure to ZnO NPs was observed by a light field microscope. Moreover, the effects of ZnO NPs on the ATP level, reactive oxygen species (ROS) generation, and apoptosis were separately explored by the DCFH-DA fluorescent probe, flow cytometry, and ATP bioluminescence assay. Moreover, the expression of cytochrome C (Cyt-C), Apaf-1, Caspase-9 and Caspase-3 at mRNA and protein levels was further determined by using quantitative PCR (Q-PCR) and western blotting. In the present study, the human peripheral blood mononuclear cells (PBMCs) were used as normal control samples for the relevant experiment. RESULTS: The results indicated that ZnO NPs could significantly inhibit human MM cell proliferation and cell death in a time- and dose-dependent manner in vitro, and this outcome can be confirmed by cell morphology and apoptosis assay. Meanwhile, the results also showed that ZnO NPs could effectively increase ROS production and decrease ATP levels in human MM cells. ZnO NPs could also significantly elevate the expression of Cyt-C, Apaf-1, Caspase-9 and Caspase-3 at mRNA and protein levels, leading to cell death. By contrast, ZnO NPs showed little cytotoxic influence on PBMCs. CONCLUSION: ZnO NPs can significantly induce human MM cell death in a time- and dose-dependent manner in vitro, decrease the ATP production and enhance the ROS generation. ZnO NPs can also increase Cyt-C, Apaf-1, Caspase-9 and Caspase-3 expression at mRNA and protein levels in human MM cells, and initiate MM cell apoptosis, indicating that Cyt-C, Apaf-1, Caspase-9 and Caspase-3 play crucial roles in ZnO NPs-induced, mitochondria-mediated apoptosis in human MM cells. Overall, ZnO NPs may be a potential agent in treating human multiple myeloma in clinical practice.


Assuntos
Morte Celular/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Nanopartículas/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Óxido de Zinco/farmacologia , Adolescente , Adulto , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Caspase 9/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Mieloma Múltiplo/metabolismo , Adulto Jovem
18.
Biomed Pharmacother ; 122: 109763, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31918288

RESUMO

Emerging evidence suggests that cinobufagin, an active ingredient in Venenum Bufonis, inhibits cell proliferation in several tumor cells. However, the anti-tumor effect of cinobufagin on nasopharyngeal carcinoma and the underlying molecular mechanisms are still unclear. In this study, we found that cinobufagin significantly inhibits the proliferation of nasopharyngeal carcinoma HK-1 cells. Further analyses demonstrated that cinobufagin induces cell cycle arrest at the S phase in HK-1 cells through downregulating the levels of CDK2 and cyclin E. Moreover, cinobufagin significantly downregulates the protein level of Bcl-2 and upregulates the levels of Bax, subsequently increasing the levels of cytoplasmic cytochrome c, Apaf-1, cleaved PARP1, cleaved caspase-3, and cleaved caspase-9, leading to HK-1 apoptosis. Furthermore, we found that cinobufagin significantly increases ROS levels and decreases the mitochondrial membrane potential in HK-1 cells. Collectively, these data imply that cinobufagin induces cell cycle arrest at the S phase and induces apoptosis through increasing ROS levels, thereby inhibiting cell proliferation in HK-1 cells. Therefore, cinobufagin is a promising bioactive agent that may contribute to the development of treatment strategies of nasopharyngeal carcinoma.


Assuntos
Apoptose/efeitos dos fármacos , Bufanolídeos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Carcinoma Nasofaríngeo/tratamento farmacológico , Neoplasias Nasofaríngeas/tratamento farmacológico , Fase S/efeitos dos fármacos , Antineoplásicos/farmacologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacos
19.
Virology ; 540: 160-164, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31928997

RESUMO

The cancer-causing Epstein-Barr virus (EBV) activates the transcription factor STAT3 upon infecting B-lymphocytes. STAT3 then activates caspase 7 to degrade cellular claspin, resulting in impaired Chk1 phosphorylation. This blockade of ATR-Chk1 signaling allows EBV-transformed cells to proliferate despite DNA lesions from virus-induced replication stress. In addressing the mechanism of caspase 7 activation, we now report that in newly-infected B-cells, STAT3 transcriptionally activates the initiator caspase, caspase 9. Caspase 9 then activates caspase 7 to impair phosphorylation of Chk1 at S345. Importantly, although cleaved products of caspase 9 are detectable in infected cells, there is simultaneous increase in the alternatively-spliced dominant-negative form of caspase 9 - and - expression of dominant-negative caspase 9 is abrogated when STAT3 activation is impaired. Thus EBV, via STAT3, activates caspase 9 but also shifts the balance of transcripts towards its dominant-negative form to allow activation of caspase 7 while avoiding death of EBV-infected cells.


Assuntos
Apoptose , Linfócitos B/metabolismo , Linfócitos B/virologia , Caspase 9/metabolismo , Transformação Celular Viral , Fator de Transcrição STAT3/metabolismo , Linfócitos B/patologia , Caspase 7/metabolismo , Caspase 9/genética , Herpesvirus Humano 4/fisiologia , Humanos , Modelos Biológicos , Fosforilação , RNA Interferente Pequeno , Fator de Transcrição STAT3/genética
20.
Environ Toxicol ; 35(5): 599-608, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31904905

RESUMO

Extensive application of amorphous silica nanoparticles (Si NPs) and ubiquitous cadmium (Cd) may increase their chances of coexposure to humans. Studies on combined effects of Si NPs and Cd in human cells are very limited. We investigated the potential mechanism of toxicity caused by coexposure of amorphous Si NPs and Cd in human liver (HepG2) cells. Results showed that Si NPs were not toxic to HepG2. However, Cd induced significant toxicity in HepG2 cells. Interestingly, we observed that a noncytotoxic concentration of Si NPs potentiated the cytotoxicity of Cd in HepG2 cells. We further noticed that coexposure of Si NPs and Cd augmented oxidative stress evidenced by the generation of oxidants (reactive oxygen species, hydrogen peroxide, and lipid peroxidation) and depletion of antioxidants (glutathione level and antioxidant enzyme activity). Coexposure of Si NPs and Cd also augmented mitochondria-mediated apoptosis in HepG2 cells indicated by altered regulation of apoptotic genes (p53, bax, bcl-2, caspase-3, and caspase-9) along with reduced mitochondrial membrane potential. Interaction data indicated that Si NPs facilitate the cellular uptake of Cd due to its strong adsorption on the surface of Si NPs. Hence, Si NPs increased the bioaccumulation and toxicity of Cd in HepG2 cells. This study warrants further research to explore the potential mechanisms of combined toxicity of Si NPs and Cd in animal models.


Assuntos
Apoptose/efeitos dos fármacos , Cádmio/toxicidade , Fígado/efeitos dos fármacos , Nanopartículas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Dióxido de Silício/toxicidade , Animais , Cádmio/administração & dosagem , Caspase 3/metabolismo , Caspase 9/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nanopartículas/administração & dosagem , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Dióxido de Silício/administração & dosagem
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