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1.
Chemosphere ; 240: 124937, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31574441

RESUMO

Nowadays, silica nanoparticles (SiNPs) as one of the most productive nano-powder, has been extensively applied in various filed. The potential harm of SiNPs has previously received severe attention. A bulk of researches have proven the adverse effect of SiNPs on the health of ecological organisms and human. However, neurotoxic impacts of SiNPs, still remain in the stage of exploration. The potential neurotoxic effects of SiNPs need to be further explored. And the toxic mechanism needs comprehensive clarification. Herein, the neurotoxicity of SiNPs of various concentrations (100, 300, 1000 µg/mL) on adult zebrafish was determined by behavioral phenotyping and confirmed by molecular biology techniques such as qPCR. Behavioral phenotype revealed observable effects of SiNPs on disturbing light/dark preference, dampening exploratory behavior, inhibiting memory capability. Furthermore, the relationship between neurotoxic symptom and the transcriptional alteration of autophagy- and parkinsonism-related genes was preliminarily assessed. Importantly, further investigations should be carried out to determine the effects of SiNPs to cause neurodegeneration in the brain as well as to decipher the specific neurotoxic mechanisms. In sum, this work comprehensively evaluated the neurotoxic effect of small-sized SiNPs on overall neurobehavioral profiles and indicated the potential for SiNPs to cause Parkinson's disease, which will provide a solid reference for the research on the neurotoxicity of SiNPs.


Assuntos
Comportamento Animal/efeitos dos fármacos , Síndromes Neurotóxicas/patologia , Dióxido de Silício/toxicidade , Peixe-Zebra/fisiologia , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Humanos , Nanopartículas/toxicidade , Peixe-Zebra/genética
2.
Chemosphere ; 238: 124647, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31466007

RESUMO

Ground water arsenic contamination is a global menace. Since arsenic may affect the immune system, leading to immunesuppression, we investigated the effects of acute arsenic exposure on the thymus and spleen using Swiss albino mice, exposed to 5 ppm, 15 ppm and 300 ppm of sodium arsenite for 7 d. Effects on cytokine balance and cell survivability were subsequently analyzed. Our data showed that arsenic treatment induced debilitating alterations in the tissue architecture of thymus and spleen. A dose-dependent decrease in the ratio of CD4+-CD8+ T-cells was observed along with a pro-inflammatory response and redox imbalance. In addition, pioneering evidences established the ability of arsenic to induce an up regulation of Hsp90, eventually resulting in stabilization of its client protein Beclin-1, an important autophagy-initiating factor. This association initiated the autophagic process, confirmed by co-immunoprecipitation assay, acridine orange staining and Western blot, indicating the effort of cells trying to survive at lower doses. However, increased arsenic assault led to apoptotic cell death in the lymphoid organs, possibly by increased ROS generation. There are several instances of autophagy and apoptosis taking place either simultaneously or sequentially due to oxidative stress. Since arsenic is a potent environmental stress factor, exposure to arsenic led to a dose-dependent increase in both autophagy and apoptosis in the thymus and spleen, and cell death could therefore possibly be induced by autophagy. Therefore, exposure to arsenic leads to serious effects on the immune physiology in mice, which may further have dire consequences on the health of exposed animals.


Assuntos
Arsênico/farmacologia , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Animais , Apoptose/efeitos dos fármacos , Arsenitos/farmacologia , Relação CD4-CD8 , Inflamação/induzido quimicamente , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Compostos de Sódio/farmacologia , Baço/efeitos dos fármacos , Baço/patologia , Timo/efeitos dos fármacos , Timo/patologia
3.
J Photochem Photobiol B ; 201: 111653, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31710929

RESUMO

Autophagy is an important process for maintaining intracellular homeostasis. Our previous study demonstrated that autophagy was down-regulated in ultraviolet B (UVB)-irradiated keratinocytes. Raffinose is a natural oligosaccharide that serves as a novel activator of autophagy and as a balancing agent to regulate the diversity of environmental stress. However, whether raffinose balances ultraviolet stress through the autophagy activation pathway has yet to be established. In this study, we found that raffinose treatment inhibited the LDH release and trypan blue staining in UVB-challenged human keratinocytes cell line HaCaT but did not affect the cleavage of apoptotic markers Caspase-3 and PARP, as well as translocation into nucleus of other cell death markers Endonuclease G and AIF. Moreover, we confirmed that raffinose treatment enhanced autophagy flux in an MTOR-independent manner in HaCaT cells. Importantly, decrease of LC3-II turnover in UVB-irradiated keratinocytes could be rescued by raffinose treatment, indicating that raffinose treatment increased autophagy in UVB-irradiated HaCaT cells. Furthermore, the effect on cell death by raffinose was inhibited when autophagy was suppressed with either a small interfering RNA targeting ATG5 (siATG5) or autophagic inhibitor wortmannin. In conclusion, we demonstrated that raffinose increases MTOR-independent autophagy and reduces cell death in UVB-irradiated keratinocytes. Our study indicated that the natural agent raffinose presents the potential value in opposing photodamage.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Rafinose/farmacologia , Raios Ultravioleta , Apoptose/efeitos da radiação , Autofagia/efeitos da radiação , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Caspase 3/metabolismo , Linhagem Celular , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo
4.
J Agric Food Chem ; 67(46): 12761-12769, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31675233

RESUMO

There is convincing evidence that consuming whole grains (WGs) may decrease the risk of colorectal cancer (CRC). Wheat bran (WB) is a rich source of dietary fiber and phytochemicals with health-promoting properties. However, the active components especially the interaction between different components in WG wheat have not been fully explored. Here, we investigated whether one of the major WB phytochemicals, alkylresorcinol (AR) C21, and the major active intestinal microbial metabolite of fiber, butyrate, could synergistically suppress human colon cancer cells. Our results demonstrated for the first time that the combination of C21 and butyrate synergistically inhibited the growth of human colon cancer cells and induced apoptosis. Further mechanistic studies demonstrated that the cotreatment of C21 and butyrate induced significant up-regulations in cleaved Poly(ADP-ribose) polymerase (PARP), cleaved caspase 3, p53 upregulated modulator of apoptosis (PUMA), cytochrome C, lipid-conjugated membrane-bound form of microtubule-associated protein 1A/1B-light chain 3 (LC3-II), and C/EBP homologous protein (CHOP) expressions, indicating the synergistic anticancer effects of C21 and butyrate were associated with induction of apoptosis, autophagy, and ER stress pathways. Notably, the C21 concentrations in the large intestinal tract of mice treated with human relevant doses of C21, were from 0.86 to 1.78 µmol/g, suggesting the C21 doses used in vitro may be achievable after daily WG wheat intake. These results provide novel insights into the dietary prevention of CRC regarding the potential interaction of bioactive WG wheat phytochemicals and the microbial metabolites of fiber.


Assuntos
Butiratos/metabolismo , Neoplasias do Colo/tratamento farmacológico , Fibras na Dieta/análise , Microbioma Gastrointestinal/efeitos dos fármacos , Intestinos/microbiologia , Compostos Fitoquímicos/administração & dosagem , Resorcinóis/administração & dosagem , Animais , Autofagia/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Neoplasias do Colo/microbiologia , Neoplasias do Colo/fisiopatologia , Humanos , Intestinos/efeitos dos fármacos , Masculino , Camundongos , Compostos Fitoquímicos/química , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Resorcinóis/química
5.
Cell Physiol Biochem ; 53(5): 747-759, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31622062

RESUMO

BACKGROUND/AIMS: Angiotensin II (Ang II) induces podocyte injury resulting in apoptosis in vitro and in vivo. However, the relationship between autophagy and apoptosis in Ang II-induced podocyte injury is unknown and the role of Ang II-induced autophagy in podocyte survival or death remains unclear. We investigated the sequential relationship between autophagy and apoptosis in Ang II-induced podocytes as well as the role of phosphatidylinositide 3-kinase (PI3-kinase). METHODS: Mouse podocytes were incubated in media containing various concentrations of Ang II and at different incubation times. The changes of podocyte autophagy and apoptosis were observed by electron microscopy, confocal imaging, western blotting, and FACS assay according to the presence of Ang II. RESULTS: Ang II enhanced the podocyte expression of the autophagic proteins, LC3A/B-II and beclin-1, and also increased the number of autophagosomes compared with control cells at early phase of 12 hours in a dose-dependent manner. This effect was inhibited by pretreatment with 3-methyladenine (3-MA), a PI3-kinase class III inhibitor. Thereafter, the Ang II-induced enhancement in autophagy decreased, whereas, podocyte apoptosis appeared later at 24 hours in concentration- and time-dependent manners in FACS and TUNEL assays. 3-MA and LY294002, a pan PI3-kinase inhibitor, further increased Ang II-induced podocyte apoptosis. Suppression of autophagy by Atg5 siRNA could induce podocyte apoptosis and further augment high-dose Ang II-induced podocyte apoptosis. CONCLUSION: These findings suggest that Ang II promotes autophagy in podocytes before apoptosis as an early adaptive cytoprotective mechanism for podocyte survival after Ang II treatment, and the transitional imbalance between autophagy and apoptosis causes podocyte injury.


Assuntos
Angiotensina II/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autofagossomos/metabolismo , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Podócitos/citologia , Podócitos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Regulação para Cima/efeitos dos fármacos
6.
Arch Virol ; 164(12): 3019-3026, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31598843

RESUMO

Polyethyleneimine (PEI) is a chemical compound that used is as a carrier in gene therapy/delivery. Some studies have investigated the microbicidal potential and antiviral activity (prophylactic or therapeutic) of PEI and its derivatives. The aim of this study was to investigate the effect of branched polyethyleneimine (bPEI) on human immunodeficiency virus (HIV) replication. Infected cells were treated with bPEI for 36 hours, and the concentration of the viral protein P24 (as a virus replication marker) was determined in cell culture supernatants. This study indicated that bPEI increased HIV replication and decreased the viability of infected cells through cytotoxicity. The toxicity of bPEI its association with and cell death (apoptosis, autophagy and necrosis) have been reported in several studies. To investigate bPEI-induced cytotoxicity, we examined apoptosis and autophagy in cells treated with bPEI, and a significant increase in HIV viral load, the P24 antigen level, autophagy, and necrosis observed. Thus, treatment with bPEI leads to cytotoxicity and higher HIV virus yield.


Assuntos
Infecções por HIV/virologia , HIV/efeitos dos fármacos , Polietilenoimina/farmacologia , Replicação Viral/efeitos dos fármacos , Autofagia/efeitos dos fármacos , HIV/genética , HIV/fisiologia , Proteína do Núcleo p24 do HIV/genética , Proteína do Núcleo p24 do HIV/metabolismo , Infecções por HIV/fisiopatologia , Humanos , Polietilenoimina/química , Carga Viral/efeitos dos fármacos
7.
Anticancer Res ; 39(10): 5403-5415, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570435

RESUMO

BACKGROUND/AIM: Tubugi-1 is a more stable and accessible synthetic counterpart of natural tubulysins. This study aimed to evaluate its cytotoxic potential against anaplastic human melanoma cells. MATERIALS AND METHODS: The viability of A-375 cells was determined by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet assay. The type of cell death and proliferative rate were investigated using flow cytometry and fluorescent microscopy, while the molecular background was evaluated by western blot. RESULTS: Tubugi-1 reduced the viability of A-375 cells, inducing massive micronucleation, followed by augmented expression of inhibitor of nuclear factor-κB and caspase-2, typical of a mitotic catastrophe. Disturbed proliferation and G2M block with prominent caspase activity, weakened the expression of B-cell lymphoma 2 and B-cell lymphoma 2-associated X transient up-regulation, coexisted with intensive autophagy. Specific inhibition of autophagy by chloroquine resulted in conversion from mitotic catastrophe to rapid apoptosis. CONCLUSION: Multilevel anticancer action of tubugi-1 is extended by co-application of an autophagy inhibitor, giving a new dimension in further preclinical advancement of this potential agent.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Citotoxinas/farmacologia , Melanoma/tratamento farmacológico , Caspase 2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Melanoma/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo
8.
Anticancer Res ; 39(10): 5417-5425, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570436

RESUMO

BACKGROUND/AIM: Chemotherapy with docetaxel (DTX) is used for castration-resistant prostate cancer (CRPC), but it is inadequate. MATERIALS AND METHODS: We evaluated the effect of the combination treatment DTX and the mTOR inhibitor temsirolimus (TEM) in the PC3 prostate cancer cell line, by focusing on the induction of autophagy and apoptosis. RESULTS: TEM induced autophagy but not apoptosis even at a high dose, whereas DTX induced apoptosis. The combination of low-dose DTX and TEM caused a 34% suppression in cell proliferation compared to monotherapy with a higher dose of DTX. The induction of apoptosis was increased by their combination. The combination with DTX overcame the induction of autophagy by TEM. The combination treatment suppressed tumor growth 72% less than the control group after 14 days of treatment in vivo. CONCLUSION: The combination of TEM and DTX induced apoptosis by overcoming autophagy and enhanced the anticancer effect compared to monotherapy.


Assuntos
Antineoplásicos/administração & dosagem , Autofagia/efeitos dos fármacos , Docetaxel/administração & dosagem , Próstata/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Sirolimo/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Terapia Combinada/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células PC-3 , Inibidores de Proteínas Quinases/administração & dosagem , Sirolimo/administração & dosagem
9.
Chem Biol Interact ; 314: 108849, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31610157

RESUMO

To provide novel insight into approaches designed to combat glioblastoma, the molecular details of the cytotoxicity of gamabufotalin, were investigated in the human glioblastoma cell line U-87. A dose-dependent cytotoxicity was observed in the cells, whereas no detectable toxicity was confirmed in mouse primary astrocytes. LDH leakage was only observed in the cells treated with a relatively high concentration (>80 ng/ml). Downregulation of the expression levels of Aurora B, cdc25A, cdc25C, cdc2, Cyclin B1 and survivin, and upregulation of the expression level of p21 were observed in treated cells and occurred in parallel with G2/M phase arrest. Treatment with gamabufotalin also downregulated the expression level of uPA, CA9, and upregulated the expression level of TIMP3, all of which are closely associated with invasion/metastasis. Autophagy induction was observed in the treated cells and the addition of wortmannin, a potent autophagy inhibitor, significantly rescued U-87 cells. These results indicate that gamabufotalin exhibits cytotoxicity against cancerous glial cells with high potency and selectivity through multiple cytotoxic signaling pathways. The activation of p38 MAPK pathway along with the upregulation of VEGF/VEGFR2 was observed in the treated cells, both of which are likely to be compensatory changes in response to gamabufotalin treatment. Intriguingly, a specific inhibitor of p38 MAPK enhanced the cytotoxicity of the drug, suggesting an important prosurvival role for p38 MAPK. We thus suggest that developing a new combination regimen of gamabufotalin plus a p38 MAPK inhibitor and/or inhibitors for VEGF/VEGFR could improve the efficacy of the drug, and may provide more therapeutic benefits to patients with glioblastoma.


Assuntos
Apoptose/efeitos dos fármacos , Bufanolídeos/farmacologia , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Autofagia/efeitos dos fármacos , Bufanolídeos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Wortmanina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
10.
J Agric Food Chem ; 67(41): 11428-11435, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31589037

RESUMO

Diosgenin and diosgenyl saponins as the major bioactive compounds isolated from dietary fenugreek seeds, yam roots, etc. possessed strong antitumor effects. To understand their detailed antitumor mechanisms, a fluorophore-appended derivative of diosgenin [Glc/CNHphth-diosgenin (GND)] was synthesized, starting from diosgenin and glucosamine hydrochloride in overall yields of 7-12% over 7-10 steps. Co-localization of GND with organelle-specific stains, transmission electron microscopy, and relative protein analyses demonstrated that GND crossed the plasma membrane through organic anion-transporting polypeptide 1B1 and distributed in the endoplasmic reticulum (ER), lysosome, and mitochondria. In this process, GND induced ER swelling, mitochondrial damage, and autophagosome and upregulating IRE-1α to induce autophagy and apoptosis. Furthermore, autophagy inhibitor chloroquine delayed the appearance of cleaved poly(ADP-ribose) polymerase and inhibited cleaved caspase 8, which indicated that GND induced autophagy to activate caspase-8-dependent apoptosis. These observations suggested that diosgenyl saponin was a potent anticancer agent that elicited ER stress and mitochondria-mediated apoptotic pathways in liver cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias Hepáticas/fisiopatologia , Extratos Vegetais/farmacologia , Saponinas/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Lisossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo
11.
Cell Physiol Biochem ; 53(4): 656-686, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31573152

RESUMO

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for approaches that identify new melanoma targets. We have previously reported a discovery of novel anti-melanoma compound 2155-14 (Onwuha-Ekpete et al., J Med Chem. 2014 Feb 27; 57(4):1599-608). In the report presented herein we aim to identify its target(s) and mechanism of action. METHODS: We utilized biotinylated analog of 2155-14 to pull down its targets from melanoma cells. Proteomics in combination with western blot were used to identify the targets. Mechanism of action of 2155-14 was determined using flow cytometry, RT-PCR, microscopy, western blot, and enzymatic activity assays. Where applicable, one-way analysis of variance (ANOVA) was used followed by Dunnett post hoc test. RESULTS: In the present study, we identified ATP-dependent RNA helicase DDX1 and heterogeneous nuclear ribonucleoproteins (hnRNPs) H1, H2 and A2/B1 as targets of anti-melanoma compound 215514. To the best of our knowledge, this is a first report suggesting that these proteins could be targeted for melanoma therapy. Mechanistic investigations showed that 2155-14 induces ER stress leading to potentiation of basal autophagy resulting in melanoma cell death in BRAF and NRAS mutated melanoma cells. CONCLUSION: Identification of mode of action of 2155-14 may provide insight into novel therapies against a broad range of melanoma subtypes. These studies were enabled by the novel probe derived from a mixture-based library, an important class of chemical biology tools for discovering novel targets.


Assuntos
Apoptose , Autofagia , RNA Helicases DEAD-box/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , Avaliação Pré-Clínica de Medicamentos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ribonucleoproteínas Nucleares Heterogêneas/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos
12.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(3): 323-327, 2019 May.
Artigo em Chinês | MEDLINE | ID: mdl-31631597

RESUMO

Objective: To determine the effects of autophagy inhibitor hydroxychloroquine (HCQ) on chemosensitivity of castration-resistant prostate cancer 22RV1 cell line in vitro and in vivo, and changes in its mRNA expressions of autophagy gene Bcelin-1, autophagy specific substrate P62 gene, pro-apoptotic gene Bax. Methods: 22RV1 cells were cultured in vitro and divided into blank control (no drug), DOC, and HCQ (20 µmol/L)+DOC groups. The concentration of DOC was set at 10 -6 mol/L, 10 -7 mol/L, and 10 -8 mol/L in the tests. Cell proliferation activities were detected by CCK-8 method 72 h after drug treatments. The 22RV1 cell suspension was injected subcutaneously into nude mice to establish transplanted tumor. The successfully modeled mice were randomly divided into three groups (five each) treated by physiological saline, DOC and HCQ+DOC (injected intraperitoneally for 4 weeks), respectively. Changes in growth of the transplanted tumor were observed. The mRNA expressions of Beclin-1, P62, and Bax were detected by qPCR. The protein expressions of Beclin-1, LC3B, and Bax were detected by Western blot. Results: In vitro: compared with the blank control, the DOC and HCQ+DOC groups showed decrease proliferation of cells( P<0.05); HCQ further lowered cell proliferation in the presence of DOC ( P<0.05), resulting in reduced half maximal inhibitory concentration (IC 50) of DOC. In vivo: compared with the model mice, the DOC and HCQ+DOC groups had decreased volume of transplanted tumor. HCQ slowed the weekly growth of tumor in the presence of DOC ( P<0.05), most obvious at the 4th week. In vitro and in vivo, HCQ+DOC upregulated the mRNA and protein expressions of Beclin-1, P62 and Bax ( P<0.05). Conclusion: HCQ can interfere with the autophagy of castration-resistant prostate cancer cells, inhibiting its proliferation and enhancing its sensitivity to chemotherapeutic drugs.


Assuntos
Autofagia/efeitos dos fármacos , Hidroxicloroquina/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Animais , Apoptose , Proteína Beclina-1/metabolismo , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Distribuição Aleatória , Proteína Sequestossoma-1/metabolismo , Proteína X Associada a bcl-2/metabolismo
13.
Chemosphere ; 235: 1050-1058, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31561294

RESUMO

Lead (Pb) is one of the most toxic heavy metals and has aroused widespread concern as it can cause severe impairments in the developing nervous system. Autophagy has been proposed as an injury factor in Pb-induced neurotoxicity. In this study, we used zebrafish embryo as a model, measured the general toxic effects of Pb, and investigated the effect of Pb exposure on autophagy, and its role in Pb-induced developmental neurotoxicity. Zebrafish embryos were exposed to Pb at concentrations of 0, 0.1, 1 or 10 µM until 4 days post-fertilization. Our data showed that exposure to 10 µM Pb significantly reduced survival rates and impaired locomotor activity. Uptake of Pb was enhanced as the concentration and duration of exposure increased. Inhibition of lysosomal degradation with bafilomycin A1 treatment abolished the suppression of Lc3-II protein expression by Pb. Furthermore, autophagosome formation was inhibited by Pb in the brain. In addition, mRNA expression of beclin1, one of the critical genes in autophagy, were decreased in Pb exposure groups at 72 h post-fertilization. Whole-mount in situ hybridization assay showed that beclin1 gene expression in the brain was reduced by Pb. Rapamycin, an autophagy inducer, partly resolved developmental neurotoxicity induced by Pb exposure. Our results suggest that autophagy plays a protective role in the developmental neurotoxicity of Pb in zebrafish embryos and larvae.


Assuntos
Autofagia/efeitos dos fármacos , Intoxicação do Sistema Nervoso por Chumbo/prevenção & controle , Chumbo/toxicidade , Peixe-Zebra/embriologia , Animais , Expressão Gênica , Larva/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo
14.
Chemosphere ; 235: 1134-1145, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31561304

RESUMO

Particulate matter (PM) from layer house has adverse effect on people and chicken respiratory health, which can further influence animal performance and reduce production efficiency. However, little study focus on the respiratory inflammation induced by PM2.5 from layer house and the underlying mechanism also unclear. In this study, human adenocarcinoma alveolar basal epithelial cells (A549 cell) was subjected to the PM2.5 from layer house to evaluate the inflammation reaction caused by PM2.5 and explore the role of Nrf2 and autophagy in regulating the inflammation. Results showed that the viability of A549 cell decreased in a time - and concentration - dependent manner after PM2.5 treatment. TNFα, IL6, and IL8 increased significantly treated with PM2.5 at 12 h. RNA sequencing indicated differentially expressed genes were enriched in immune system process, oxidative stress (OS), endoplasmic reticulum stress (ERS), and autophagy. Further studies showed TLR4 - NFκB p65 signal pathway involved in the inflammation reaction caused by PM2.5. The overexpression of Nrf2 decreased the level of TNFα, IL6, IL8 markedly as well as the level of NFκB p65 and NFκB pp65. OS and ERS were also limited under overactivation of Nrf2 in PM2.5 treated cells. Autophagy induced by PM2.5 promoted the inflammation through increasing the level of NFκB p65 and NFκB pp65. Autophagy deficient strengthened the expression of Nrf2. Collectively, our study revealed Nrf2 prevents inflammation caused by layer house PM2.5 stimulation, however, autophagy exerts a promotive role in TLR4 - NFκB p65 mediating inflammation in A549 cell.


Assuntos
Autofagia/fisiologia , Inflamação/etiologia , Fator 2 Relacionado a NF-E2/fisiologia , Material Particulado/efeitos adversos , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo , Células A549 , Animais , Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Humanos , Inflamação/induzido quimicamente , Inflamação/prevenção & controle , Fator 2 Relacionado a NF-E2/farmacologia , Estresse Oxidativo/genética , Transdução de Sinais
15.
Chem Biol Interact ; 314: 108839, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31563593

RESUMO

Here, we show that incubation of three human gastrointestinal cancer cell lines (HCT15, LoVo and MKN45) with doxorubicin (DOX) provokes autophagy through facilitating production of reactive oxygen species (ROS). HCT15 cell treatment with DOX resulted in up-regulation of Beclin1, down-regulation of Bcl2, activation of AMPK and JNK, and Akt inactivation, all of which were restored by pretreating with an antioxidant N-acetyl-l-cysteine. These data suggest that all the autophagy-related alterations evoked by DOX result from the ROS production. In the DOX-resistant cancer cells, degree of autophagy elicited by DOX was milder than the parental cells, and DOX treatment hardly activated the ROS-dependent apoptotic signals [formation of 4-hydroxy-2-nonenal (HNE), cytochrome-c release into cytosol, and activation of JNK and caspase-3], inferring an inverse correlation between cellular antioxidant capacity and autophagy induction by DOX. Monitoring of expression levels of aldo-keto reductases (AKRs) in the parental and DOX-resistant cells revealed an up-regulation of AKR1B10 and/or AKR1C3 with acquiring the DOX resistance. Knockdown and inhibition of AKR1B10 or AKR1C3 in these cells enhanced DOX-elicited autophagy. Measurement of DOX-reductase activity and HNE-sensitivity assay also suggested that both AKR1B10 (via high HNE-reductase activity) and AKR1C3 (via low HNE-reductase and DOX-reductase activities) are involved in the development of DOX resistance. Combination of inhibitors of autophagy and the two AKRs overcame DOX resistance and cross-resistance of gastrointestinal cancer cells with resistance development to DOX or cis-diamminedichloroplatinum. Therefore, concomitant treatment with the inhibitors may be effective as an adjuvant therapy for elevating DOX sensitivity of gastrointestinal cancer cells.


Assuntos
Aldo-Ceto Redutases/metabolismo , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Aldeído Redutase/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Gastrointestinais/metabolismo , Neoplasias Gastrointestinais/patologia , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacos
16.
Aquat Toxicol ; 215: 105286, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31479757

RESUMO

Chlorpyrifos (CPF) is an environmental pollutant with increasing importance due to its high toxicity to fish and aquatic animals. In the present study, we divided 120 common carp (Cyprinus carpio L.) into two groups including control group and CPF group, CPF group was exposed to 14.5 µg/L CPF for 30 d. 17 miRNAs were differentially expressed in CPF group head kidney tissues according to the results of miRNAome analysis. In addition, histopathological examination and electron microscopy proved that CPF exposure could lead to damage of head kidney and obvious apoptosis characteristics. The possible target genes of miRNA were predicted using online target gene prediction websites, miRNAome sequencing, GO and KEGG enrichment. miRNAome results showed that expression of miR-731 and miR-2188-3p in CPF group was 0.48 time and 0.45 time as control group, respectively. qRT-PCR results proved the reality of miRNAome. During CPF exposure, mRNA expression of TLR pathway genes and its downstream genes involved in autophagy and apoptosis pathway including TLR1, TLR2, TLR7, TLR9, MyD88, IRAK1, IRAK4, IRF7, PI3K, AKT, mTOR, Caspase3, Caspase8 and Bax were differentially increased under CPF exposure, along with ATG13 and Bcl2 decreased at the same time. Western blot results indicated that apoptosis related protein Caspase3 and Caspase8 were differentially up-regulated in the CPF group. In summary, CPF exposure could induce apoptosis while inhibited autophagy in head kidney of common carp via the regulation of miR-2188-3p and miR-731 by targeting TLR pathway. These results provide new insights for unveiling the biological effects of CPF and miRNAs in common carp.


Assuntos
Apoptose/genética , Carpas/genética , Clorpirifos/toxicidade , Rim Cefálico/lesões , MicroRNAs/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Autofagia/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Rim Cefálico/efeitos dos fármacos , Rim Cefálico/metabolismo , Rim Cefálico/ultraestrutura , MicroRNAs/química , MicroRNAs/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade
17.
Life Sci ; 235: 116842, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31494170

RESUMO

MicroRNAs plays important role in the development of myocardial infarction (MI). The aim of this study was to analyze whether miR-429 has effect on the process of autophagy in myocardial anoxia/reoxygenation (AR) or ischemia/reperfusion (IR) injury and explore the underlying mechanism. The results showed that miR-429 was significantly decreased in MI mouse hearts and AR treated cardiomyocytes. Dual luciferase activity assay proved that MO25 was the direct target of miR-429. MO25 was dramatically decreased in AR treated cardiomyocytes. Overexpression of miR-429 dramatically decreased the expression of MO25, whereas inhibition of miR-429 noticeably increased the expression of MO25. In addition, overexpression of miR-429 reduced GFP-LC3B labelled cells, decreased the number of vesicle and autophagosome in each cardiomyocyte, and induced cell apoptosis in AR treated cardiomyocytes. In contrast, inhibition of miR-429 had the opposite effect. The further in vivo study showed that when mouse in IR group were injected with antagomiR-429, the weight of left ventricular was increased and infarct size was significantly decreased. Finally, both the in vitro and in vivo study showed that the expression of MO25, LKB1, pAMPKa, ATG13, p62 and LC3BI/II was noticeably increased by antagomiR-429. In conclusion, our results suggested that antagonism of miR-429 ameliorates anoxia/reoxygenation injury in cardiomyocytes by enhancing MO25/LKB1/AMPK mediated autophagy.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Hipóxia/metabolismo , MicroRNAs/antagonistas & inibidores , Miócitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Autofagossomos/efeitos dos fármacos , Contagem de Células , Vesículas Citoplasmáticas/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Camundongos , MicroRNAs/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Traumatismo por Reperfusão Miocárdica , Miocárdio/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo
18.
Exp Parasitol ; 206: 107730, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31494215

RESUMO

Phospholipids are the main component of membranes and are responsible for cell integrity. Alkylphospholipid analogues (APs) were first designed as antitumoral agents and were later tested against different cell types. Trypanosoma cruzi, the Chagas disease etiological agent, is sensitive to APs (edelfosine, miltefosine and ilmofosine) in vitro. We investigated the effect of synthetic ring substituted AP against epimastigotes, amastigotes and trypomastigotes. TCAN26, could inhibit the in vitro growth of epimastigotes and amastigotes with the 50% inhibitory concentrations (IC50) in the nanomolar range. Trypomastigotes lysis was also induced with 24-h treatment and a LC50 of 2.3 µM. Ultrastructural analysis by electron microscopy demonstrated that TCAN26 mainly affected the parasite's membranes leading to mitochondrial and Golgi cisternae swelling, membrane blebs, and autophagic figures in the different parasite developmental stages. While the Golgi of the parasites was significantly affected, the Golgi complex of the host cells remained normal suggesting a specific mechanism of action. In summary, our results suggest that TCAN 26 is a potent and selective inhibitor of T. cruzi growth probably due to disturbances of phospholipid biosynthesis.


Assuntos
Adamantano/farmacologia , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Adamantano/química , Animais , Antiprotozoários/química , Antiprotozoários/farmacologia , Autofagia/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Complexo de Golgi/efeitos dos fármacos , Concentração Inibidora 50 , Dose Letal Mediana , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Mitocôndrias/efeitos dos fármacos , Fosforilcolina/química , Tripanossomicidas/química , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/ultraestrutura
19.
Int J Nanomedicine ; 14: 6249-6268, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31496684

RESUMO

Purpose: To develop an intravesical instillation system for the treatment of bladder cancer, rapamycin (Rap) was encapsulated into liposomes and then homogeneously dispersed throughout a poloxamer 407 (P407)-based hydrogel. Methods: Rap-loaded conventional liposomes (R-CL) and folate-modified liposomes (R-FL) were prepared using a film hydration method and pre-loading technique, and characterized by particle size, drug entrapment efficiency, and drug loading. The cellular uptake behavior in folate receptor-expressing bladder cancer cells was observed by flow cytometry and confocal laser scanning microscopy using a fluorescent probe. In vitro cytotoxic effects were evaluated using MTT assay, colony forming assay, and Western blot. For in vivo intravesical instillation, Rap-loaded liposomes were dispersed in P407-gel, generating R-CL/P407 and R-FL/P407. Gel-forming capacities and drug release were evaluated. Using the MBT2/Luc orthotopic bladder cancer mouse model, in vivo antitumor efficacy was evaluated according to regions of interest (ROI) measurement. Results: R-CL and R-FL were successfully prepared, at approximately <160 nm, 42% entrapment efficiency, and 57 µg/mg drug loading. FL cellular uptake was enhanced over 2-fold than that of CL; folate receptor-mediated endocytosis was confirmed using a competitive assay with folic acid pretreatment. In vitro cytotoxic effects increased dose-dependently. Rap-loaded liposomes inhibited mTOR signaling and induced autophagy in urothelial carcinoma cells. With gelation time of <30 seconds and gel duration of >12 hrs, both R-CL/P407 and R-FL/P407 preparations transformed into gel immediately after instillation into the mouse bladder. Drug release from the liposomal gel was erosion controlled. In orthotopic bladder cancer mouse model, statistically significant differences in ROI values were found between R-CL/P407 and R-FL/P407 groups at day 11 (P=0.0273) and day 14 (P=0.0088), indicating the highest tumor growth inhibition by R-FL/P407. Conclusion: Intravesical instillation of R-FL/P407 might represent a good candidate for bladder cancer treatment, owing to its enhanced retention and FR-targeting.


Assuntos
Ácido Fólico/química , Hidrogéis/química , Sirolimo/administração & dosagem , Sirolimo/farmacologia , Temperatura Ambiente , Administração Intravesical , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Coloides , Modelos Animais de Doenças , Liberação Controlada de Fármacos , Feminino , Receptores de Folato com Âncoras de GPI/metabolismo , Humanos , Lipossomos , Camundongos , Tamanho da Partícula , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico
20.
J Cancer Res Clin Oncol ; 145(10): 2507-2517, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31485766

RESUMO

BACKGROUND: Autophagy plays an important role in regulating cisplatin (CDDP) resistance in gastric cancer cells. However, the underlying mechanism of methioninase (METase) in the regulation of autophagy and CDDP resistance of gastric cancer cells is still not clear. MATERIALS AND METHODS: Western blot was used to detect the levels of autophagy-related proteins, multidrug-resistant 1 (MDR-1), and FoxM1 protein. LncRNA HULC was detected by qRT-PCR. Cell viability was detected using CCK-8 assay. The interaction between lncRNA HULC and FoxM1 was confirmed by RNA pull-down and RIP assay. RESULTS: Lentiviral vector carrying METase (LV-METase) suppressed autophagy and CDDP resistance of drug-resistant gastric cancer cells. LncRNA HULC was significantly downregulated in drug-resistant gastric cancer cells transfected with LV-METase. Besides, we found that lncRNA HULC interacted with FoxM1. In addition, METase suppressed autophagy to reduce CDDP resistance of drug-resistant gastric cancer cells through regulating HULC/FoxM1, and interfering HULC suppressed autophagy to reduce CDDP resistance of drug-resistant gastric cancer cells through regulating FoxM1. Finally, interfering HULC inhibited tumor growth in vivo. CONCLUSION: METase suppressed autophagy to reduce CDDP resistance of drug-resistant gastric cancer cells through regulating HULC/FoxM1 pathway.


Assuntos
Autofagia/efeitos dos fármacos , Autofagia/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Forkhead Box M1/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Longo não Codificante/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Cisplatino/farmacologia , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Ligação Proteica , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
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