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1.
Biomed Pharmacother ; 123: 109812, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31945696

RESUMO

Wu-tou decoction (WTD), a classic Traditional Chinese medicine formula, has been extensively used in the treatment of neuropathic pain (NP) such as chronic inflammatory pain, trigeminal neuralgia, and cancer-induced pain. Our previous studies have shown that the severity of mechanical allodynia and thermo hypersensitivity in NP rats are reduced by WTD, of which analgesic candidates are paeoniflorin (Pae) and liquiritin (Liq). The aim of this study was to clarify the molecular mechanisms of WTD, Pae and Liq against NP based on the primary rat glial cells in vitro. The gene expression levels of neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and Artemin and C-C chemokine receptor type 5 (CCR5) were augmented by inflammatory cytokines, while chemokines increased only CCR5 gene expression. The constitutive and cytokine-augmented neurotrophic factor gene expression was enhanced by WTD, Pae, and Liq through PI3K- and PKA-dependent pathways in rat glial cells, leading to the increase of NGF and BDNF production. Furthermore, the CCR5 gene expression under basal and chemokine-treated conditions was suppressed by these reagents, in which signal pathway(s) was independent on the activation of PI3K and PKA. Moreover, there was no cytotoxicity in the WTD, Pae, and Liq treatments in glial cells. Thus, these results provide a novel evidence that WTD may exert the anti-NP actions by predominantly increasing the production of neurotrophic factors through PI3K- and PKA-signaling pathways in rat glial cells. Furthermore, Pae and Liq may play as analgesic candidates in WTD-mediated NP management.

2.
J Ethnopharmacol ; 249: 112385, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31730888

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Cerebral ischemia, also known as stroke, can stimulate the proliferation and migration of endogenous neural stem cells (NSCS) in subventricular zone of the lateral ventricle and subgranularzone of the dentate gyrus in the adult hippocampus as a defense response to damage. However, the proliferation of endogenous NSCS is insufficient for central nervous system repair. Neurogenesis and anti-neuroinflammation are two important aspects for neuroprotection. Rhizome Ligusticum chuanxiong (LC), the dried rhizomes of Ligusticum striatum DC., has been widely used to treat stroke for over hundreds of years in Traditional Chinese Medicine. PURPOSE: of the study: Previous reports on pharmacological mechanism of LC mainly focus on the cerebral blood flow and thrombolysis. We aim to explore whether LC provides neuroprotective effect by increasing neurogenesis and inhibiting the IL-1ß, TNF-α and expressions of glial fibrillary acidic protein. MATERIALS AND METHODS: LC extract was delivered to microsphere-embolized (ME) cerebral ischemia Wister rats to examine its neuroprotection. Body weight, neurological scores, hematoxylin-eosin staining (HE), TUNEL assay were conducted for neurological damage. Neurogenesis was evaluated by assessing the expression of Doublecortin (DCX) and neurogenic differentiation1 (NeuroD1) through immunofluorescence staining. Western blot performed to measure the protein levels of growth associated protein-43(GAP-43), glial fibrillary acidic protein (GFAP). IL-1ß and TNF-α was detected by Elisa. RESULTS: LC alleviated pathomorphological change and apoptosis of neurons in the hippocampus caused by ME surgery. Furthermore, LC significantly increased the DCX in the DG of adult rat hippocampus at 14 days after surgery. A significant upregulation of GAP-43 compared to the ME after LC was administered. Besides, LC decreased pro-inflammatory cytokine (IL-1ß, TNF-α) and protein level of GFAP. CONCLUSION: The finding suggested that LC had the ability to protect neurons by promoting the endogenous proliferation of neuroblast and production of neural differentiation factor in rats after ischemia injury. Meanwhile, LC can anti-neuroinflammation, which is important for the treatment of neuron injury. Accordingly, LC perhaps a promising medicine for neuron damage therapy after cerebral ischemia.

3.
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
4.
Int J Mol Sci ; 20(20)2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31640144

RESUMO

Progranulin (PGRN) plays a crucial role in diverse biological processes, including cell proliferation and embryonic development. PGRN can be cleaved by neutrophil elastase to release granulin (GRN). PGRN has been found to inhibit inflammation. Whereas, GRN plays a role as a pro-inflammatory factor. However, the pathophysiological roles of PGRN and GRN, at early stages after cerebral ischemia, have not yet been fully understood. The aim of this study was to obtain further insight into the pathologic roles of PGRN and GRN. We demonstrated that the amount of PGRN was significantly increased in microglial cells after cerebral ischemia in rats and that neutrophil elastase activity was also increased at an early stage after cerebral ischemia, resulting in the production of GRN. The inhibition of neutrophil elastase activity suppressed PGRN cleavage and GRN production, as well as the increase in pro-inflammatory cytokines, after cerebral ischemia. The administration of an elastase inhibitor decreased the number of injured cells and improved the neurological deficits test scores. Our findings suggest that an increase in the activity of elastase to cleave PGRN, and to produce GRN, was involved in an inflammatory response at the early stages after cerebral ischemia, and that inhibition of elastase activity could suppress the progression of cerebral ischemic injury.

5.
Sci Rep ; 9(1): 11782, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409872

RESUMO

The N-methyl-D-aspartate (NMDA) receptor has been implicated in several neurodegenerative diseases, including stroke. Low-density lipoprotein receptor-related protein 1 (LRP1) plays pivotal roles in endocytosis and signaling in the cell. Immature LRP1 is processed by furin in the trans-Golgi network (TGN) and transported to the cell surface as its mature form. Activation of mature LRP1 exerts a protective effect against glutamate-induced degeneration of the rat retinal ganglion cells, as was shown in our previous study. However, the roles of LRP1 in the pathogenesis of excitotoxic neuronal injuries remain to be determined. The aim of this present study was to achieve further insight into the pathophysiologic roles of LRP1 after excitotoxic neuronal injuries. Our findings are the first to demonstrate that LRP1 was significantly cleaved by furin after cerebral ischemia in rats as well as after exposure of cultured cortical neurons to NMDA. It was noteworthy that the intracellular domain (ICD) of LRP1 was co-localized with TGN and furin. Furthermore, a furin inhibitor inhibited the cleavage of LRP1 and co-localization of LRP1-ICD with TGN or furin. Our findings suggest that furin-mediated cleavage of LRP1 and changes in the localization of LRP1-ICD were involved in the excitotoxic neuronal injury.

6.
BMC Complement Altern Med ; 19(1): 216, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31412862

RESUMO

BACKGROUND: Breast cancer is still the most common malignant tumor that threatens the female's life in the world, especially triple-negative breast cancer (TNBC), one of the most difficult subtypes. Lack of targeted therapies brings about urgent demand for novel treatments. In this study we aim to investigate the anti-tumor activity of Berberine (BBR), a Chinese plant-derived alkaloid, against the TNBC cell line MDA-MB-231 and elucidate its mechanism referring to anti-inflammation. METHODS: Cell inhibition rate was measured by Cell Proliferation Assay, the cytotoxic effects was detected by Lactate dehydrogenase (LDH) leakage assay, the colony formation and migration potential were evaluated by colony formation assay and wound healing assay, the release of inflammatory cytokines was detected by EMD multifactor detection, and alterations of proteins and genes related to the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway were analyzed using western blotting and real-time Polymerase Chain Reaction (PCR). RESULTS: BBR reduce the viability of MDA-MB-231 cells and increased the release of LDH from the cells in a dose-dependent manner, with and inhibition of colony formation potential and migration of the cells. BBR also caused a marked reduction in the secretion of proinflammatory cytokines, Interleukin-1α (IL-1α), Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Besides, a down-regulated behavior was observed with the expression of P2X purinoceptor 7 (P2X7), NLRP3, pro-caspase-1, apoptosis-associated speck-like protein containing a caspase-activation and recruitment domain (ASC), caspase-1 p20, Interleukin-18 (IL-18), IL-1ß proteins and NLRP3, Caspase-1 and ASC mRNAs in the NLRP3 inflammasome cascade. CONCLUSIONS: Our results confirmed that BBR can effectively affect both tumor outgrowth and spontaneous metastasis in TNBC, and that we identified a new mechanism associated with inhibition the NLRP3 inflammasome pathway, suggesting its potential therapeutic relevance in clinical use.


Assuntos
Berberina/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Inflamassomos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Neoplasias de Mama Triplo Negativas/imunologia , Apoptose/efeitos dos fármacos , Caspase 1/genética , Caspase 1/imunologia , Feminino , Humanos , Inflamassomos/genética , Inflamassomos/imunologia , Interleucina-18/genética , Interleucina-18/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/fisiopatologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
7.
J Ethnopharmacol ; 243: 112122, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31356965

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Toad skin came from Bufo bufo gargarizans Cantor and Bufo melanostictus Schneider. As the traditional Chinese medicine, it had the effect of clearing away heat and detoxification. In traditional applications, toad skin was often used for the treatment of cancer and inflammation. Total indolealkylamines (IAAs) from this medicine were proved the main compounds exert anti-inflammatory activity in our previous research. AIM OF THE STUDY: In the present study, we aimed to investigate the potential mechanism of anti-inflammatory activity of IAAs on LPS induced zebrafish. MATERIALS AND METHODS: LPS induced zebrafish was applicated as an in vivo inflammation model to clarify the structure-activity relationship of 4 major IAAs (N-methyl serotonin, bufotenine, dehydrobufotenine and bufothionine) from toad skin. Quantitative RT-PCR was applied to detect key cytokines and members of the MyD88-dependent signaling pathway. In addition, the targeted lipidomics was conducted to find out the potential biomarkers in the inflammatory zebrafish. Network pharmacology was used to unveil the main enzymes closely related to the target lipids. RESULTS: Our results showed that the anti-inflammatory activity of free IAAs (N-methyl serotonin, bufotenine and dehydrobufotenine) was more potent than that of combined IAAs (bufothionine). RT-PCR demonstrated that 4 IAAs exerted antiendotoxin inflammatory effect via suppressing the TLR4/MyD88/NF-κB and TLR4/MyD88/MAPKs signaling pathway. A total of 33 possible inflammatory biomarkers, including 14 SM, 6 Cer, 11 PC and 2 GlcCer, triggered by LPS were screened out. The levels of most of candidates could be regulated toward a normal level by IAAs, especially in N-methyl serotonin and dehydrobufotenine groups. Enzymes especially LBP, PLA2, CERK, SMPD and SGMS were found closely associated with the regulation of most lipid markers. CONCLUSIONS: Overall, the mechanism underlying the anti-inflammatory activity of IAAs probably attributed to their capability to suppress NF-κB and MAPKs inflammatory pathway. Meanwhile, IAAs could also interfere the metabolism of SM, Cer and PC probably by regulating LBP, PLA2, CERK, SMPD and SGMS.


Assuntos
Anti-Inflamatórios/farmacologia , Indóis/farmacologia , Animais , Animais Geneticamente Modificados , Anti-Inflamatórios/química , Bufonidae , Citocinas/genética , Feminino , Indóis/química , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/genética , Larva , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipopolissacarídeos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Fator 88 de Diferenciação Mieloide/genética , NF-kappa B/metabolismo , Pele , Receptor 4 Toll-Like/genética , Peixe-Zebra
8.
Am J Chin Med ; 47(5): 1149-1170, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31311297

RESUMO

Three-dimensionally (3D) cultured tumor cells (spheroids) exhibit more resistance to therapeutic agents than the cells cultured in traditional two-dimensional (2D) system (monolayers). We previously demonstrated that arsenic disulfide (As2S2) exerted significant anticancer efficacies in both 2D- and 3D-cultured MCF-7 cells, whereas 3D spheroids were shown to be resistant to the As2S2 treatment. L-buthionine-(S, R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, has been regarded to be a potent candidate for combinatorial treatment due to its GSH modulation function. In the present study, we introduced BSO in combination with As2S2 at a low concentration to investigate the possible enhancing anticancer efficacy by the combinatorial treatment on 2D- and 3D-cultured MCF-7 cells. Our results presented for the first time that the combination of As2S2 and BSO exerted potent anticancer synergism in both MCF-7 monolayers and spheroids. The IC50 values of As2S2 in combinatorial treatment were significantly lower than those in treatment of As2S2 alone in both 2D- and 3D-cultured MCF-7 cells (P<0.01, respectively). In addition, augmented induction of apoptosis and enhanced cell cycle arrest along with the regulation of apoptosis- and cell cycle-related proteins, as well as synergistic inhibitions of PI3K/Akt signals, were also observed following co-treatment of As2S2 and BSO. Notably, the combinatorial treatment significantly decreased the cellular GSH levels in both 2D- and 3D-cultured MCF-7 cells in comparison with each agent alone (P<0.05 in each). Our results suggest that the combinatorial treatment with As2S2 and BSO could be a promising novel strategy to reverse arsenic resistance in human breast cancer.


Assuntos
Antineoplásicos/farmacologia , Arsenicais/farmacologia , Neoplasias da Mama/fisiopatologia , Butionina Sulfoximina/farmacologia , Sulfetos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Técnicas de Cultura de Células , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Células MCF-7 , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo
9.
Mol Neurobiol ; 56(3): 1946-1956, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29981053

RESUMO

We previously demonstrated that proliferation of endogenous neural progenitor cells is enhanced by cerebral ischemia and that phosphatidylinositol 3-kinase (PI3-K)/Akt-dependent glycogen synthase kinase (GSK)-3ß signaling is involved in ischemia-induced neurogenesis. It is important to learn more about the regulation of proliferation and differentiation of neural progenitor cells under ischemic conditions, as such knowledge that may serve as the basis for the development of new therapeutic approaches for stroke. However, it remains to be addressed whether a change in that signaling pathway is induced in neural progenitor cells. We prepared neural progenitor cells by using the neurosphere method and conducted experiments to determine the relative contributions of the PI3-K/Akt-dependent GSK-3ß signaling pathway to the proliferation and differentiation of neural progenitor cells under the hypoxic condition in vitro. We showed that hypoxic exposure induced the proliferation of neural progenitor cells. This proliferation was accompanied by phosphorylation of Akt and GSK-3ß at its Ser9. Furthermore, treatment with a PI3-K inhibitor decreased the hypoxia-induced phosphorylation of GSK-3ß and proliferation of neural progenitor cells. Furthermore, hypoxic exposure enhanced the differentiation of neural progenitor cells, and this increased differentiation was not affected by treatment with the PI3-K inhibitor. Although the expression of NeuroD1 mRNA during cell differentiation was also enhanced by hypoxic exposure, this increased expression was not affected by treatment with the PI3-K inhibitor. Our findings suggest that the PI3K/Akt-dependent GSK-3ß signaling pathway was involved in the proliferation of neural progenitor cells under a pathologic condition, such as hypoxia and/or cerebral ischemia in vivo.


Assuntos
Hipóxia Celular/fisiologia , Proliferação de Células/fisiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Células-Tronco Neurais/citologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos
10.
Oncol Rep ; 41(1): 27-42, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30320388

RESUMO

In the present study, the antitumor effects of arsenic disulfide (As2S2) on the proliferative, survival and migratory ability of human breast cancer MCF­7 and MDA­MB­231 cells were investigated, and its potential underlying molecular mechanisms with an emphasis on cell cycle arrest, apoptosis induction, autophagy induction and reactive oxygen species (ROS) generation were determined. The results indicated that As2S2 significantly inhibited the viability, survival and migration of breast cancer cells in a dose­dependent manner. In addition, it was identified that As2S2 induced cell cycle arrest primarily at G2/M phase in the two breast cancer cell lines by regulating the expression of associated proteins, including cyclin B1 and cell division cycle protein 2. In addition to cell cycle arrest, As2S2 also triggered the induction of apoptosis in cells by activating the expression of pro­apoptotic proteins, including caspase­7 and ­8, as well as increasing the B­cell lymphoma 2 (Bcl­2)­associated X protein/Bcl­2 ratio, while decreasing the protein expression of anti­apoptotic B­cell lymphoma extra­large. In addition, As2S2 stimulated the accumulation of microtubule­associated protein 1A/1B­light chain 3 (LC3)­II and increased the LC3­II/LC3­I ratio, indicating the occurrence of autophagy. As2S2 treatment also inhibited the protein expression of matrix metalloproteinase­9 (MMP­9), but increased the intracellular accumulation of ROS in the two breast cancer cell lines, which may assist in alleviating metastasis and attenuating the progression of breast cancer. Taken together, the results of the present study suggest that As2S2 inhibits the progression of human breast cancer cells through the regulation of cell cycle arrest, intrinsic and extrinsic apoptosis, autophagy, MMP­9 signaling and ROS generation.


Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Arsenicais/farmacologia , Neoplasias da Mama/metabolismo , Sulfetos/farmacologia , Apoptose , Autofagia , Neoplasias da Mama/tratamento farmacológico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Espécies Reativas de Oxigênio/metabolismo
11.
Molecules ; 24(1)2018 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-30591646

RESUMO

Toad skin and toad venom, as two kinds of Chinese medicine, are prepared from Bufo bufo gargarizans Cantor and Bufo melanostictus Schneider. However, they display distinct properties in traditional application, and the hydrophilic ingredients are the possible distinguishing chemicals between them. In this work, 36 and 22 hydrophilic components were characterized from toad skin and venom, respectively, by UHPLC-HR-MS/MS, including amino acids, nucleosides, polypeptides, and indolealkylamines (IAAs). Among them, 15 compounds were unambiguously confirmed by comparison with standards. The CID-MS/MS fragmentation behaviors of seven indolealkylamine references were investigated to ascertain three types of structures. Subsequently, 11 high abundance contents of hydrophilic ingredients were determined from 11 batches of toad skin and 4 batches of toad venom by UPLC-QqQ-MS/MS. The quantitative results showed that the content of main IAAs in toad venom was much higher than in skin. In addition, the N-methyl serotonin (free IAA), bufothionine (combined IAA), and total IAAs sample were selected for anti-inflammatory evaluation in lipopolysaccharide (LPS) stimulated zebrafish embryo models. The obvious anti-inflammatory activities of IAAs were observed, especially for the free IAAs. This study illustrated IAAs were the main distinct hydrophilic components that probably lead to the difference between toad skin and toad venom in traditional applications.


Assuntos
Aminas/farmacologia , Venenos de Anfíbios/química , Anti-Inflamatórios/farmacologia , Bufonidae/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Pele/química , Espectrometria de Massas em Tandem/métodos , Aminas/química , Animais , Cromatografia Líquida de Alta Pressão , Alcaloides Indólicos/farmacologia , Lipopolissacarídeos , Análise de Componente Principal , Compostos de Quinolínio/farmacologia , Padrões de Referência , Serotonina/análogos & derivados , Serotonina/farmacologia , Peixe-Zebra
12.
Int J Oncol ; 53(6): 2488-2502, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30272276

RESUMO

Glioblastoma is the most common and lethal intracranial tumor type, characterized by high angiogenic and infiltrative capacities. To provide a novel insight into therapeutic strategies against glioblastoma, the cytotoxicity of arenobufagin and hellebrigenin was investigated in the human glioblastoma cell line, U-87. Similar dose-dependent cytotoxicity was observed in the cells, whereas no detectable toxicity was confirmed in mouse primary astrocytes. Treatment with each drug downregulated the expression levels of Cdc25C, Cyclin B1 and survivin, which occurred in parallel with G2/M phase arrest. Necrotic-like cell death was only observed in the cells treated with a relatively high concentration (>100 ng/ml). These results indicate that the two drugs exhibited distinct cytotoxicity against cancerous glial cells with high potency and selectivity, suggesting that growth inhibition associated with G2/M phase arrest and/or necrosis were attributed to their toxicities. Activation of the p38 mitogen activated protein kinase (MAPK) signaling pathway was also observed in treated cells. Notably, a specific inhibitor of p38 MAPK, SB203580, itself caused a significant decrease in cell viability, and further enhanced the cytotoxicity of the two drugs, suggesting an important pro-survival role for p38 MAPK. Given that p38 MAPK serves an essential role in promoting glioblastoma cell survival, developing a novel combination regimen of arenobufagin/hellebrigenin plus a p38 MAPK inhibitor may improve the efficacy of the two drugs, and may provide more therapeutic benefits to patients with glioblastoma. The qualitative assessment demonstrated the existence of arenobufagin in the cerebrospinal fluid of arenobufagin-treated rats, supporting its clinical application.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Bufanolídeos/farmacologia , Ciclo Celular/efeitos dos fármacos , Glioblastoma/metabolismo , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclina B1/genética , Ciclina B1/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Piridinas/farmacologia , Ratos , Survivina/genética , Survivina/metabolismo , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismo
13.
Cancer Cell Int ; 18: 113, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30123091

RESUMO

Background: Triple-negative breast cancer (TNBC) is one of the most difficult subtypes of breast cancer to treat due to its aggressive, metastatic behavior, and a lack of a targeted therapy. Trivalent arsenic derivatives (arsenite, AsIII) with remarkable clinical efficacy in acute promyelocytic leukemia has been demonstrated to exhibit inhibitory effect against breast cancer cells. To provide novel insight into the development of new therapeutic strategies, antitumor activity of AsIII and tetrandrine (Tetra), a Chinese plant-derived alkaloid, against the TNBC cell line MDA-MB-231 in vitro and in vivo was investigated. Methods: Cytotoxicity was evaluated using cell viability, lactate dehydrogenase leakage and cell cycle assay. Alterations of genes related to cell proliferation and death were analyzed using western blotting. In vivo antitumor activity of AsIII alone or in combination with Tetra was studied using MDA-MB-231 xenografts in nude mice. Results: Synergistic cytotoxic effects of two drugs were observed in the cells. In vivo study also showed that co-administration of AsIII and Tetra significantly reduced tumor volume and weight, directly supporting its in vitro antitumor activity. No deaths and reduction of body-weight were observed after a long-term co-administration, indicating its good tolerability. S-phase arrest associated with the upregulation of FOXO3a, p27 along with decreased Cyclin D1 expression was observed in the cells treated with the combined regimen. A substantial upregulated p21 expression and downregulated phospho-FOXO3a and Cyclin D1 expression was observed in the tumor tissues of mice co-administered with AsIII and Tetra. Autophagy induction was observed in the combination treatment in vitro and in vivo. The addition of wortmannin, a potent autophagy inhibitor, significantly rescued MDA-MB-231 cells from their cytotoxicity of AsIII and Tetra. Conclusions: S-phase arrest, autophagic and necrotic cell death contribute to the cytocidal effects of the combined regimen of AsIII and Tetra. Considering our previous study showing synergistic cytotoxic effects of the combined regimen in estrogen receptor-positive breast cancer cell line MCF-7, these results suggest that development of the combination regimen of AsIII plus Tetra may offer many benefits to patients with different types of breast cancer.

14.
Chem Biol Interact ; 294: 9-17, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30125548

RESUMO

Among five major anthocyanin compounds, delphinidin exhibited the most potent and selective cytocidal effect against HL-60, a trivalent arsenic (As(III))-resistant cell line. Co-treatment with delphinidin and As(III) resulted in the reduction of IC50 value for As(III) from 11.2 to 1.5 µM, which was considered as clinically achieved concentrations of As(III). The combination treatment strongly preferred to selectively enhance the cytotoxicity of As(III) against HL-60 cells rather than human peripheral blood mononuclear cells. The induction of apoptosis as evidenced by the increase of sub-G1 cells, DNA fragmentation, annexin V-positive cells and the activation of caspase-8, -9 and -3 was observed in HL-60 cells co-treated with As(III) and delphinidin. Similar to the activation pattern of caspases, a substantial decrease in the expression level of Bid along with the loss of mitochondrial membrane potential was also observed. These results suggested that the combination treatment triggered a convergence of the intrinsic and extrinsic pathways of apoptosis via the activation of caspase-8 and cleaved Bid. Delphinidin itself significantly decreased the intracellular GSH ([i]GSH) and nuclear factor-κB (NF-κB) binding activity, and further returned As(III)-triggered increment of [i]GSH and enhancement of NF-κB binding activity to control level. Additionally, buthionine sulfoximine, a GSH depletor; JSH-23, a NF-κB inhibitor, also mimicked the capacity of delphinidin to significantly induce the reduction of [i]GSH along with the potentiation of As(III) cytotoxicity in HL-60 cells. These observations suggested that delphinidin-induced sensitization of HL-60 cells to As(III) was caused by the reduction of [i]GSH, which was probably associated with the inhibitory effect of delphinidin on NF-κB binding activity. These findings further suggest that delphinidin-induced sensitization of HL-60 cells to As(III) may lead to dose reduction of As(III) in clinical application, and ultimately contribute to minimizing its side effects.


Assuntos
Antocianinas/química , Apoptose/efeitos dos fármacos , Arsenitos/farmacologia , Antocianinas/farmacologia , Arsenitos/química , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Sinergismo Farmacológico , Glutationa/metabolismo , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , NF-kappa B/química , NF-kappa B/metabolismo
15.
Int J Oncol ; 52(6): 1959-1971, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29620191

RESUMO

In China, arsenic disulfide (As2S2) has been used for the treatment of hematological malignancies. The present study aimed to evaluate the effects of As2S2 on the human breast cancer MCF­7 cell line cultured in both two­dimensional (2D) monolayers and three­dimensional (3D) spheroids to explore its therapeutic potential in breast cancer treatment. Cellular viability and the induction of apoptosis were examined with a cell counting kit­8 (CCK­8) assay and flow cytometric analysis, respectively. Alterations in the expression levels of apoptosis­associated proteins, including Bcl­2­associated X protein (Bax), B­cell lymphoma 2 (Bcl­2), p53, and caspase­7, as well as the cell survival­associated proteins, phosphatidylinositol 3­kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR), were assessed by western blotting. Although a dose­dependent reduction in cell viability, which occurred in association with the induction of apoptosis triggered by the addition of 2­24 µM As2S2, was observed in both 2D­ and 3D­culture systems, 3D spheroids were less sensitive to the cytotoxic effect of As2S2 compared with the 2D cultured cells. A significant increase in the expression levels of Bax, p53, and caspase­7 was observed in treated 2D­cultured cells, whereas a similar increase in the expression levels of Bax was only confirmed in treated 3D spheroids, although there was a trend towards the increased expression of p53 and caspase­7 in the 3D spheroids. These results suggested that these molecules are closely associated with As2S2­mediated cytotoxicity in the two culture systems, and further suggested that the difference in the sensitivity to As2S2 between 2D monolayers and 3D spheroids may be attributed to the differential alterations in the expression levels of proteins associated with cell mortality. Significant downregulation of the expression levels of Bcl­2, PI3K, Akt and mTOR was observed in the two culture systems. Taken together, the results of the present study demonstrated that As2S2 inhibits cell viability and induces apoptosis in both 2D­ and 3D­ cultured MCF­7 cells, which may be associated with activation of the pro­apoptotic pathway and the inhibition of pro­survival signaling. These results have provided novel insights into clinical applications of As2S2 in the treatment of patients with breast cancer.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Arsenicais/farmacologia , Neoplasias da Mama/metabolismo , Técnicas de Cultura de Células/métodos , Esferoides Celulares/efeitos dos fármacos , Sulfetos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo
16.
Arch Biochem Biophys ; 648: 53-59, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29678629

RESUMO

Cell adhesive biomaterials have been used for various cells in culture, especially for primary cultures of neurons. Here we examined laminin-111 and its active peptides conjugated to chitosan matrices (ChtMs) for primary culture of rat cortical neurons. Laminin-111 on poly-d-lysine substrate promoted neuronal cell attachment and differentiation. The biological activity of six active laminin-111-derived peptides was examined using a peptide-ChtM construct. When the syndecan-binding peptides, AG73 (RKRLQVQLSIRT, mouse laminin α1 chain 2719-2730) and C16 (KAFDITYVRLKF, laminin γ1 chain 139-150), were conjugated to chitosan, AG73-ChtM and C16-ChtM showed potent neuronal cell attachment activity and promoted axon extension by primary cultured rat cortical neurons. However, the remaining peptides, including integrin-binding peptides, did not show activity when conjugated to ChtM. AG73-ChtM and C16-ChtM also supported neuron survival for at least 4 weeks in serum-free medium without a glia feeder layer. These data suggest that AG73-ChtM and C16-ChtM are useful for primary cultures of central nervous system neurons and have a potential for use as functional biomaterials for tissue engineering in the central nervous system.


Assuntos
Encéfalo/citologia , Quitosana/química , Quitosana/farmacologia , Laminina/química , Neurônios/citologia , Neurônios/efeitos dos fármacos , Peptídeos/química , Sequência de Aminoácidos , Animais , Adesão Celular/efeitos dos fármacos , Camundongos , Neuritos/efeitos dos fármacos , Ratos
17.
Am J Cancer Res ; 8(3): 366-386, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29636995

RESUMO

Arsenic disulfide, a major effective component of realgar, has been investigated for its anti-cancer potential and shown to have therapeutic efficacies in hematological and some solid tumors. However, its effect against breast cancer is rarely reported. In this study, we investigated the anti-cancer effects of As2S2 in human breast cancer cell lines MCF-7 and MDA-MB-231, and further elucidated its underlying mechanisms. As2S2 significantly inhibited cell viabilities, induced apoptosis, and led to cell cycle arrest in both cell lines with a dose- and time-dependent manner. As2S2 upregulated pro-apoptotic proteins like p53 and PARP in MCF-7 cells. Besides, As2S2 downregulated anti-apoptotic proteins like Bcl-2 and Mcl-1, as well as cell cycle-related proteins cyclin A2 and cyclin D1 in both cell lines. Of note, the expression level of cyclin B1 was downregulated in MCF-7 cells, whereas, upregulated in MDA-MB-231 cells. Moreover, As2S2 significantly inhibited the pro-survival signals in PI3K/Akt pathway in both cell lines. In conclusion, As2S2 inhibited cell viabilities, induced apoptosis and cell cycle arrest in both MCF-7 and MDA-MB-231 cell lines by regulating the expression of key proteins involved in related pathways. These results provide fundamental insights into the clinical application of As2S2 for treatment of patients with breast cancer.

18.
Sci Rep ; 8(1): 5212, 2018 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-29581474

RESUMO

The proprotein convertases (PCs) act as serine proteases and are known to convert diverse precursor proteins into their active forms. Among the PCs, furin has been considered to play a crucial role not only in embryogenesis, but also in the initiation and progression of certain pathologic conditions. However, the roles played by furin with respect to neuronal cell injuries remain to be determined. An excessive influx of Ca2+ through the N-methyl-d-aspartate (NMDA) receptor has been associated with diverse neurological and neurodegenerative disorders. The aim of this study was to achieve further insight into the pathophysiologic roles of furin in cultured cortical neurons. We demonstrated that furin inhibitors dose-dependently prevented neuronal injury induced by NMDA treatment. Neuronal injury induced by NMDA treatment was attenuated by the calpain inhibitor calpeptin. And the increase observed in the activity of calpain after NMDA treatment was significantly inhibited by these furin inhibitors. Furthermore, calpain-2 activity, which was evaluated by means of the immunoblotting assay, was increased by NMDA treatment. It was noteworthy that this increased activity was almost completely inhibited by a furin inhibitor. Our findings suggested that furin is involved in NMDA-induced neuronal injury by acting upstream of calpain.


Assuntos
Calpaína/genética , Furina/genética , Doenças Neurodegenerativas/genética , Neurônios/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/genética , Animais , Morte Celular/genética , Desenvolvimento Embrionário/genética , Furina/antagonistas & inibidores , Humanos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Pró-Proteína Convertases/genética , Ratos
19.
Anticancer Res ; 38(4): 2101-2108, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29599328

RESUMO

BACKGROUND/AIM: Chemo-sensitivity of two-dimensional (2D) monolayers and three-dimensional (3D) spheroids of human breast cancer MCF-7 cells were investigated. MATERIALS AND METHODS: MCF-7 cells were cultured in monolayers or spheroids established using a thermo-reversible gelatin polymer, in the presence of daunorubicin, docetaxel, or As2S2 Cell proliferation was examined by a Cell Counting Kit-8 assay. RESULTS: Daunorubicin, docetaxel, and As2S2 dose-dependently decreased the MCF-7 cell proliferation in both 2D- and 3D-culture systems. The 3D spheroids were less sensitive to these agents than the 2D cultured cells. Verapamil, an inhibitor of P-glycoprotein, partially enhanced the antiproliferative effects of the agents. DL-buthionine-(S, R)-sulfoximine significantly increased (p<0.05), while N-acetyl-L-cysteine significantly inhibited the antiproliferative effects of As2S2 (p<0.003). CONCLUSION: The 3D spheroids showed less sensitivity to the antiprolliferative efficacies of anticancer agents than the 2D cultured cells. P-Glycoprotein is suggested to be partially implicated in drug resistance. Reduction of cellular glutathione level enhanced the As2S2 cytotoxicity.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Técnicas de Cultura de Células/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Esferoides Celulares/patologia , Arsenicais/farmacologia , Proliferação de Células/efeitos dos fármacos , Daunorrubicina/farmacologia , Docetaxel , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Células MCF-7 , Esferoides Celulares/efeitos dos fármacos , Sulfetos/farmacologia , Taxoides/farmacologia , Tecidos Suporte/química
20.
Sci Rep ; 8(1): 2459, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29410497

RESUMO

Thalidomide was originally used as a sedative and found to be a teratogen, but now thalidomide and its derivatives are widely used to treat haematologic malignancies. Accumulated evidence suggests that thalidomide suppresses nerve cell death in neurologic model mice. However, detailed molecular mechanisms are unknown. Here we examined the molecular mechanism of thalidomide's neuroprotective effects, focusing on its target protein, cereblon (CRBN), and its binding protein, AMP-activated protein kinase (AMPK), which plays an important role in maintaining intracellular energy homeostasis in the brain. We used a cerebral ischemia rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). Thalidomide treatment significantly decreased the infarct volume and neurological deficits of MCAO/R rats. AMPK was the key signalling protein in this mechanism. Furthermore, we considered that the AMPK-CRBN interaction was altered when neuroprotective action by thalidomide occurred in cells under ischemic conditions. Binding was strong between AMPK and CRBN in normal SH-SY5Y cells, but was weakened by the addition of H2O2. However, when thalidomide was administered at the same time as H2O2, the binding of AMPK and CRBN was partly restored. These results suggest that thalidomide inhibits the activity of AMPK via CRBN under oxidative stress and suppresses nerve cell death.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Proteases Dependentes de ATP/genética , Isquemia Encefálica/tratamento farmacológico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Talidomida/farmacologia , Complexos Ubiquitina-Proteína Ligase/genética , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Proteases Dependentes de ATP/antagonistas & inibidores , Proteases Dependentes de ATP/metabolismo , Animais , Isquemia Encefálica/enzimologia , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Reposicionamento de Medicamentos , Regulação da Expressão Gênica , Humanos , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Imunossupressores/farmacologia , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/patologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/patologia , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/metabolismo
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