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1.
Small ; 16(8): e1907598, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32003943

RESUMO

Lightweight and mechanically strong protein fibers are promising for many technical applications. Despite the widespread investigation of biological fibers based on spider silk and silkworm proteins, it remains a challenge to develop low-cost proteins and convenient spinning technology for the fabrication of robust biological fibers. Since there are plenty of widely available proteins in nature, it is meaningful to investigate the preparation of fibers by the proteins and explore their biomedical applications. Here, a facile microfluidic strategy is developed for the scalable construction of biological fibers via a series of easily accessible spherical and linear proteins including chicken egg, quail egg, goose egg, bovine serum albumin, milk, and collagen. It is found that the crosslinking effect in microfluidic chips and double-drawn treatment after spinning are crucial for the formation of fibers. Thus, high tensile strength and toughness are realized in the fibers, which are comparable or even higher than that of many recombinant spider silks or regenerated silkworm fibers. Moreover, the suturing applications in rat and minipig models are realized by employing the mechanically strong fibers. Therefore, this work opens a new direction for the production of biological fibers from natural sources.

2.
BMC Cancer ; 19(1): 949, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615459

RESUMO

BACKGROUND: In the quest for new anti-cancer drugs, the drug discovery process has shifted to screening of active ingredients in traditional eastern medicine. Matrine is an active alkaloid isolated from plants of the Sophora genus used in traditional Chinese herbal medicine that exhibits a wide spectrum of biological properties and has a potential as an anti-proliferative agent. In this study, we investigated the anticancer property of MASM, ([(6aS, 10S, 11aR, 11bR, 11cS)210-Methylamino-dodecahydro-3a, 7a-diaza-benzo (de)anthracene-8-thione]), a potent derivative of matrine. METHODS: Four epithelial cancer cell lines representing the dominant cancers, namely: A549 (non-small-cell lung cancer cell line), MCF-7 and MDA-MB-231 (breast cancer cell lines), and Hela (cervical cancer cell line) were employed, and the mechanistic underpinning of MASM-induced apoptosis was investigated using flow cytometry, western blot and immunofluorescence. RESULTS: MASM, induced apoptosis via caspase 3 dependent and independent pathways, and autophagy in all the four cancer cell lines, but post-EMT (epithelial mesenchymal transition) cells showed greater sensitivity to MASM. Scavenging reactive oxygen species using N-acetylcysteine rescued all cancer cell lines from apoptosis and autophagy. Mechanistic analysis revealed that MASM induced autophagy involves inhibition of Akt signaling and the activation of Erk and p38 signaling, and inhibition of autophagy further enhanced the apoptosis induced by MASM. CONCLUSIONS: These results indicate that MASM possesses potency against cancer cells and modulating autophagy during MASM administration could be used to further enhance its therapeutic effects.


Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Sistema de Sinalização das MAP Quinases , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinolizinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Células A549 , Alcaloides/química , Antineoplásicos/química , Sobrevivência Celular/efeitos dos fármacos , Descoberta de Drogas/métodos , Medicamentos de Ervas Chinesas/química , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células HeLa , Humanos , Células MCF-7 , Neoplasias/patologia , Quinolizinas/química , Transdução de Sinais/efeitos dos fármacos , Sophora/química
3.
Folia Histochem Cytobiol ; 57(2): 64-73, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31246264

RESUMO

INTRODUCTION: This study endeavors to analyze the effects of miR-1204 on the expression of DEK oncogene in non-small cell lung cancer (NSCLC) cell lines and to study the molecular mechanisms of these effects. MATERIAL AND METHODS: The miR-1204 mimics and inhibitors were transfected into the (A549 and SPC) NSCLC cells. Then the mRNA levels, cell viability, apoptosis rate, morphology and caspase activity were determined. The expression of apoptosis-related proteins Bcl-2 and Bax was also analyzed. RESULTS: In NSCLC cell lines (A549 and SPC), DEK mRNA levels were down-regulated in miR-1204 overex-pression group. In miR-1204 inhibition group, the expression of DEK mRNA showed an opposite trend. The overexpression of miR-1204 increases the apoptosis rate in NSCLC cells. The Bcl-2 levels in the miR-1204 over-expression group were decreased, while the Bax level was increased. In the miR-1204 inhibition group, expression of Bcl-2 and Bax showed opposite trends. Cell staining revealed cell's morphological changes; the apoptosis in the miR-1204 overexpression group revealed significant morphological features, such as brighter nuclei and nu-clear condensation. Results indicated a typical characteristic of apoptosis in the miR-1204 overexpression group. Caspase-9 and Caspase-3 were involved in the apoptosis pathway, which was mediated by miR-1204 and DEK. CONCLUSIONS: The miR-1204 induces apoptosis of NSCLC cells by inhibiting the expression of DEK. The mech-anism of apoptosis involves down-regulation of Bcl-2 and up-regulation of Bax expression. Moreover, the apoptosis was mediated by mitochondria-related caspase 9/3 pathway.


Assuntos
Apoptose/fisiologia , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas Cromossômicas não Histona/genética , Neoplasias Pulmonares/genética , MicroRNAs/fisiologia , Proteínas Oncogênicas/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Mensageiro/fisiologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regulação para Cima , Proteína X Associada a bcl-2/metabolismo
4.
ACS Chem Biol ; 14(3): 516-525, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30789695

RESUMO

As a host defense peptide, hymenochirin-1B has attracted increasing attention for its strong cytotoxic activities. However, its poor selectivity and proteolytic stability remain major obstacles for clinical application. To solve these problems, we designed and synthesized a series of peptide analogues of hymenochirin-1B based on cationic residue substitution and stapling combined with a glycosylation strategy. Some analogues showed improvement not only in selectivity and proteolytic stability but also in antitumor activity. Among them, the glycosylated stapled peptide H-58 was identified as the most potential antitumor peptide. Flow cytometry and a competitive binding assay revealed that H-58 displayed significant antitumor selectivity. Confocal microscopy and nuclear staining with Hoechst dye demonstrated that H-58 entered the nucleus and caused DNA damage. In summary, the strategy of glycosylated stapled peptides is a promising approach for improving the antitumor selectivity, proteolytic stability, and antitumor activity of hymenochirin-1B, which can be used for other bioactive peptide modifications.


Assuntos
Acetilglucosamina/química , Peptídeos Catiônicos Antimicrobianos/química , Antineoplásicos/química , Aminoácidos/química , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Glicosilação , Humanos , Estrutura Molecular , Estabilidade Proteica , Proteólise , Relação Estrutura-Atividade
5.
Chem Sci ; 10(5): 1522-1530, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30809370

RESUMO

Two major pharmacological hurdles severely limit the widespread use of small peptides as therapeutics: poor proteolytic stability and membrane permeability. Importantly, low aqueous solubility also impedes the development of peptides for clinical use. Various elaborate side chain stapling chemistries have been developed for α-helical peptides to circumvent this problem, with considerable success in spite of inevitable limitations. Here we report a novel peptide stapling strategy based on the dithiocarbamate chemistry linking the side chains of residues Lys(i) and Cys(i + 4) of unprotected peptides and apply it to a series of dodecameric peptide antagonists of the p53-inhibitory oncogenic proteins MDM2 and MDMX. Crystallographic studies of peptide-MDM2/MDMX complexes structurally validated the chemoselectivity of the dithiocarbamate staple bridging Lys and Cys at (i, i + 4) positions. One dithiocarbamate-stapled PMI derivative, DTCPMI, showed a 50-fold stronger binding to MDM2 and MDMX than its linear counterpart. Importantly, in contrast to PMI and its linear derivatives, the DTCPMI peptide actively traversed the cell membrane and killed HCT116 tumor cells in vitro by activating the tumor suppressor protein p53. Compared with other known stapling techniques, our solution-based DTC stapling chemistry is simple, cost-effective, regio-specific and environmentally friendly, promising an important new tool for the development of peptide therapeutics with improved pharmacological properties including aqueous solubility, proteolytic stability and membrane permeability.

6.
Adv Sci (Weinh) ; 5(7): 1800234, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30027052

RESUMO

A robust, microwave-assisted, highly efficient, solid-phase peptide synthesis method for preparing isopeptide-linked 62-mer and 76-mer isoubiquitins and polyubiquitin is developed. The strategy avoids the use of costly resins and pseudoprolines, and the isopeptide-linked building blocks can be assembled with high initial purity within 1 day. All seven diubiquitins are successfully synthesized on a multi-milligram scale; a four-segment, three-ligation method is used to obtain a K33-/K11-linked mixed triubiquitin in excellent yield. Circular dichroism and crystallographic analyses are used to verify the structures of the well-folded, synthetic polyubiquitin chains. The facile synthetic strategy is expected to be generally applicable for the rapid synthesis of isopeptide-linked isoUbs and to pave the way for the study of longer polyubiquitin chains.

7.
Oncol Lett ; 15(6): 8573-8581, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29844811

RESUMO

DEK is a protein ubiquitously expressed in multicellular organisms as well as certain unicellular organisms. It is associated with the regulation of cell proliferation, differentiation, migration, apoptosis, senescence, self-renewal and DNA repairing. In tumor cells it is associated with the carcinogenesis process, however there have been few previous studies into the expression of DEK in lung cancer. In the present study the expression level of DEK mRNA and protein was detected in lung cancer tissues and non-cancerous counterparts by performing reverse transcription-quantitative polymerase chain reaction and immunohistochemical staining. It was revealed that the expression of DEK was increased in lung cancer tissues compared with normal tissue. Knock-down and over-expression of DEK in A549 cells were performed to determine the role of DEK in tumor formation. An MTT assay, colony formation assay and Matrigel invasion assay demonstrated that DEK positively regulated cell proliferation and invasion. These results suggest that DEK is highly expressed in lung cancer tissues and positively regulates cell proliferation and invasion.

8.
Appl Biochem Biotechnol ; 185(1): 357, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29637397

RESUMO

The original version of this article unfortunately contained a mistake in the image of Figure 7.

9.
Eur J Med Chem ; 145: 661-672, 2018 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-29348072

RESUMO

Osteoporosis is a metabolic bone disease characterized by low bone mass and micro-architectural deterioration of bone, for which the underlying mechanism is an imbalance between bone resorption and bone remodeling. The protein-protein interactions between receptor activator of nuclear factor-κB ligand (RANKL), RANK (its receptor), and osteoprotegerin (OPG), are known to mediate the development and activation of osteoclasts in bone remodeling, and are regarded as a pivotal therapeutic target for the treatment of osteoporosis. Herein, we disclose the successful development of a novel glycopeptide (OM-2), the structure of which is based on the key interacting sites of the reported RANKL and OPG crystal structure. OM-2 exhibited potent binding affinity with RANKL and resistance to degradation by protease enzymes. It also blocked RANKL/RANK interactions, and inhibited osteoclastogenesis in vitro. In vivo studies confirmed that OM-2 could effectively reduce bone loss and inhibit osteoclast activation in ovariectomized (OVX) mice at a dosage of 20.0 mg/kg/day. Accordingly, OM-2 is suggested as a therapeutic candidate for postmenopausal osteoporosis (PMOP) and osteoclastogenesis-related diseases like rheumatoid arthritis (RA). More importantly, its identification validates our structure-based strategy for the development of drugs that target the RANKL/RANK/OPG system.


Assuntos
Glicopeptídeos/farmacologia , Osteoporose/tratamento farmacológico , Osteoprotegerina/farmacologia , Ovariectomia , Ligante RANK/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Animais , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Relação Dose-Resposta a Droga , Feminino , Glicopeptídeos/síntese química , Glicopeptídeos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Estrutura Molecular , Osteoporose/metabolismo , Osteoprotegerina/química , Ligação Proteica/efeitos dos fármacos , Ligante RANK/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade
10.
Mol Cell Biochem ; 441(1-2): 63-76, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28887716

RESUMO

Non-small-cell lung cancer (NSCLC) is still the main threat of cancer-associated death. Current treatment of NSCLC has limited effectiveness, and unfortunately, the prognosis of NSCLC remains poor. Therefore, a novel strategy for cancer therapy is urgently needed. Stem cell therapy has significant potential for cancer treatment. Mesenchymal stem cells (MSCs) with capacity for self-renewal and differentiation into various cells types exhibit the feature of homing to tumor site and immunosuppression, have been explored as a new treatment for various cancers. Studies revealed that the broad repertoire of trophic factors secreted by MSCs extensively involved in the interplay between MSCs and tumor cells. In this study, we confirmed that MSCs do have the paracrine effect on proliferation and migration of NSCLC cells (A549, NCI-H460, and SK-MES-1). Co-culture system and conditioned medium experiments results showed that soluble factors secreted by MSCs inhibited the proliferation of NSCLC cells in vitro. The scratch assay showed that conditioned medium of MSCs could suppress the migration of NSCLC cells in vitro. Western blot results showed that the expression of proteins relevant to cell proliferation, anti-apoptosis, and migration was remarkably decreased via MAPK/eIF4E signaling pathway. We speculated that soluble factors secreted by MSCs might be responsible for inhibitory mechanism of NSCLC cells. By Human Gene Expression Microarray Assay and recombinant Vascular Endothelial Growth Factor 165 (VEGF165) neutralizing experiment, we verified that VEGF might be responsible for the down-regulation of proteins related to cell proliferation, anti-apoptosis, and migration by suppressing translation initiation factor eIF4E via MAPK signaling pathway. Taken together, our study demonstrated that a possible trophic factor secreted by MSCs could manipulate translation initiation of NSCLC cells via MAPK signaling pathway, and significantly affect the fate of tumor cells, which will be a new strategy for cancer therapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Movimento Celular , Proliferação de Células , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases , Células-Tronco Mesenquimais/metabolismo , Comunicação Parácrina , Células A549 , Carcinoma Pulmonar de Células não Pequenas/patologia , Técnicas de Cocultura , Células HEK293 , Humanos , Neoplasias Pulmonares/patologia , Células-Tronco Mesenquimais/patologia
11.
Appl Biochem Biotechnol ; 184(1): 212-227, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28664526

RESUMO

Mesenchymal stem cells (MSCs) exhibit the feature of homing to tumor site and being immunosuppressive, which have broad prospects in tumor therapy. However, MSCs are commonly cultured in a two-dimensional (2D) condition, which would gradually loss some in vivo important properties. In this study, we built a three-dimensional (3D) system with collagen/Matrigel scaffolds to culture MSCs. The results indicated that MSCs in 3D scaffolds showed higher proliferation ability than that of in 2D cells. In vitro, 3D-cultured MSC-conditioned media (CM) significantly inhibited the proliferation of hepatoma cells HepG2 than that of in 2D-cultured MSC-CM and control groups. In vivo, animal transplantation experiment showed that the treatment of 3D-cultured MSC-CM could further significantly delay the tumor initiation and decrease the tumor volume. The microarray, quantitative PCR, and ELISA assay found that MSCs cultured in the 3D system expressed and secreted more amounts of IL-24. RT-PCR and western blot results showed that IL-24 can activate JAK1-STAT3 pathway via IL22R1 and IL20R2, and further inhibit the proliferation of HepG2 cells. Taken together, these results demonstrated that MSCs cultured in the 3D system had an inhibitory effect on the proliferation of HepG2 cells, probably through secreting more IL-24, which activated JAK1-STAT3 signaling and finally inhibited the cell proliferation to delay tumor initiation. This study also provided a simpler and more reliable approach for MSCs to suppress tumor cells, and provided effective experimental data for clinical treatment of tumor and experimental basis.


Assuntos
Células-Tronco Mesenquimais/citologia , Animais , Sequência de Bases , Western Blotting , Técnicas de Cultura de Células , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Células HEK293 , Células Hep G2 , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Reação em Cadeia da Polimerase em Tempo Real
12.
Int Immunopharmacol ; 54: 254-260, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29169044

RESUMO

Esculentoside A (EsA), a saponin isolated from Phytolacca esculenta, is reported as a potent suppressor of pro-inflammatory functions of macrophages. However, little is known about the target proteins of EsA for its anti-inflammatory activity. In the present study, to identify the intracellular target for EsA, affinity resins bearing immobilized EsA were used to capture binding proteins of EsA from RAW264.7 cell lysates. Mass spectrography and Western blot analysis of captured proteins indicated that ribosomal protein S3a preferentially bound to EsA affinity resin. Competition experiment further demonstrated that free EsA can disturb the specific interaction between recombinant RPS3a and affinity resin. Surface Plasmon Resonance analysis confirmed that EsA directly bound to RPS3a. Lentivirus-mediated RNAi RPS3a resulted in suppression of TNF-α and IL-6 production and impediment of signal transduction in LPS-stimulated RAW264.7 cells, indicating that RPS3a is required for LPS-triggered signaling during induction of pro-inflammatory cytokines. In addition, EsA inhibited the expression of inflammatory factors more strongly in the case of RPS3a interference. These results suggest that EsA exerts its anti-inflammatory activity by targeting RPS3a and impairing its signaling function. These new findings not only extended our understanding on the intracellular mechanisms of EsA, but also indicated RPS3a as an essential component for LPS-mediated pro-inflammatory signaling, thus implying RPS3a as a novel therapeutic target for anti-inflammatory therapy.


Assuntos
Anti-Inflamatórios/metabolismo , Macrófagos/imunologia , Ácido Oleanólico/análogos & derivados , Proteínas Ribossômicas/metabolismo , Saponinas/metabolismo , Animais , Humanos , Interleucina-6/metabolismo , Lipopolissacarídeos/imunologia , Camundongos , Ácido Oleanólico/metabolismo , Phytolaccaceae/imunologia , Ligação Proteica , Células RAW 264.7 , RNA Interferente Pequeno/genética , Proteínas Ribossômicas/genética , Transdução de Sinais , Ressonância de Plasmônio de Superfície , Fator de Necrose Tumoral alfa/metabolismo
13.
Chem Biodivers ; 15(1)2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29125222

RESUMO

As natural-product-derived antibiotics, desotamides A - D and wollamides exhibit growth inhibitory activity against Gram-posivite bacteria (IC50 0.6 - 7 µm) and are noncytotoxic to mammalian cells (IC50  > 30 µm). Herein we firstly report the total synthesis of above two cyclohexapeptides as well as a series of structural variants through solid phase peptide synthesis, of which 3 displayed a 2-fold increase of antibacterial activity when compared with the original peptide 1. This strategy may offer good improvements for the synthesis of other cyclic peptides.


Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Bactérias Gram-Positivas/crescimento & desenvolvimento , Células Hep G2 , Humanos , Células MCF-7 , Testes de Sensibilidade Microbiana , Estrutura Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Relação Estrutura-Atividade
14.
Chem Sci ; 8(11): 7368-7373, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29163887

RESUMO

The all-hydrocarbon peptide stapling strategy has recently been extensively explored in drug discovery. There remains the potential for improvement regarding the retention of the amino acid side chains at the stapled positions. Herein, we describe a new series of amino acids that not only contain the native side chains, but also carry the alkenyl arms that are needed for the ring-closing stapling chemistry. We incorporate the new amino acids into a ß-catenin-binding domain of Axin (469-482) and develop a new category of stapled peptides with the retention of the native side chains. These stapled peptides exhibit high α-helicity, strong proteolytic stability and good cell permeability. Biochemical experiments demonstrate that these stapled peptides can activate ß-catenin more efficiently than canonical stapled peptides due to the presence of extra side chains. We expect that the new side-chain-retention stapling method would expand the scope of the all-hydrocarbon stapled peptide strategy by retaining some important peripheral residues for protein-protein interactions or preserving key hydrophilic side chains to improve solubility.

15.
Bioorg Med Chem Lett ; 27(20): 4678-4681, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28916339

RESUMO

According to the previously reported potent dual l-peptide PMI of p53-MDM2/MDMX interactions, a series of d-amino acid mutational PMI analogues, PMI-1-4, with enhanced proteolytic resistence and in vitro tumor cell inhibitory activities were reported, of which Liposome-PMI-1 showed a stronger inhibitory activity against the U87 cell lines than Nutlin-3. This d-amino acid mutation strategy may give a hand for enhancing the potential of peptide drugs.


Assuntos
Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Dicroísmo Circular , Humanos , Lipossomos/química , Simulação de Dinâmica Molecular , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores
16.
Cell Death Dis ; 8(9): e3037, 2017 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-28880271

RESUMO

Postmenopausal osteoporosis (POMP) is a public health problem characterized by decreased bone density and increased fracture risk. Over-activated osteoclastogenesis plays a vital role in POMP. Here we developed a novel bioactive compound MASM (M19) based on sophocarpine. Although it showed no significant effects on osteogenesis and adipogenesis for bone marrow-derived mesenchymal stem cells (BMSCs) in vitro, it could significantly inhibit RANKL/M-CSF induced osteoclastogenesis through suppressing NF-κB, MAPKs and PI3K/Akt pathways in vitro and ameliorate bone loss in ovariectomized mice in vivo. Ribosomal protein s5 (RPS5) has been identified as a target of M19 and regulates PI3K/Akt, NF-κB and MAPKs pathways in osteoclastogenesis. Overexpressions of RPS5 synergistically inhibited osteoclastogenesis with M19 while silencing RPS5 compromised M19 inhibitory effects on osteoclastogenesis in vitro. Among the three pathways, Akt plays a major role in M19 effects. The Akt activator SC79 partially reversed the inhibitory effects on osteoclastogenesis by M19 and RPS5-knocking-down. It indicates that RPS5 serves as a potential candidate target for inhibiting osteoclastogenesis and osteoporosis therapy and M19 is a promising agent for POMP treatment.


Assuntos
Alcaloides/farmacologia , Conservadores da Densidade Óssea/farmacologia , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteoporose Pós-Menopausa/tratamento farmacológico , Proteínas Ribossômicas/genética , Acetatos/farmacologia , Alcaloides/síntese química , Animais , Benzopiranos/farmacologia , Densidade Óssea/efeitos dos fármacos , Conservadores da Densidade Óssea/síntese química , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Fator Estimulador de Colônias de Macrófagos/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese/genética , Osteoporose Pós-Menopausa/etiologia , Osteoporose Pós-Menopausa/genética , Osteoporose Pós-Menopausa/patologia , Ovariectomia/efeitos adversos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/agonistas , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante RANK/antagonistas & inibidores , Ligante RANK/farmacologia , Proteínas Ribossômicas/agonistas , Proteínas Ribossômicas/metabolismo , Transdução de Sinais
17.
FASEB J ; 31(11): 4855-4865, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28739641

RESUMO

Osteoporosis is a metabolic bone disease characterized by decreased bone density and strength due to excessive loss of bone protein and mineral content. The imbalance between osteogenesis by osteoblasts and osteoclastogenesis by osteoclasts contributes to the pathogenesis of postmenopausal osteoporosis. Estrogen withdrawal leads to increased levels of proinflammatory cytokines. Overactivated osteoclasts by inflammation play a vital role in the imbalance. Matrine is an alkaloid found in plants from the Sophora genus with various pharmacological effects, including anti-inflammatory activity. Here we demonstrate that matrine significantly prevented ovariectomy-induced bone loss and inhibited osteoclastogenesis in vivo with decreased serum levels of TRAcp5b, TNF-α, and IL-6. In vitro matrine significantly inhibited osteoclast differentiation induced by receptor activator for NF-κB ligand (RANKL) and M-CSF in bone marrow monocytes and RAW264.7 cells as demonstrated by tartrate-resistant acid phosphatase (TRAP) staining and actin-ring formation as well as bone resorption through pit formation assays. For molecular mechanisms, matrine abrogated RANKL-induced activation of NF-κB, AKT, and MAPK pathways and suppressed osteoclastogenesis-related marker expression, including matrix metalloproteinase 9, NFATc1, TRAP, C-Src, and cathepsin K. Our study demonstrates that matrine inhibits osteoclastogenesis through modulation of multiple pathways and that matrine is a promising agent in the treatment of osteoclast-related diseases such as osteoporosis.-Chen, X., Zhi, X., Pan, P., Cui, J., Cao, L., Weng, W., Zhou, Q., Wang, L., Zhai, X. Zhao, Q., Hu, H., Huang, B., Su, J. Matrine prevents bone loss in ovariectomized mice by inhibiting RANKL-induced osteoclastogenesis.


Assuntos
Alcaloides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases , Osteoclastos/metabolismo , Osteoporose Pós-Menopausa/metabolismo , Quinolizinas/farmacologia , Ligante RANK/biossíntese , Animais , Catepsina K/metabolismo , Feminino , Humanos , Interleucina-6/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/patologia , Osteoporose Pós-Menopausa/tratamento farmacológico , Osteoporose Pós-Menopausa/patologia , Ovariectomia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células RAW 264.7 , Fosfatase Ácida Resistente a Tartarato/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Quinases da Família src/metabolismo
19.
Protein Sci ; 26(5): 1037-1048, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28257598

RESUMO

The potential for infection by coronaviruses (CoVs) has become a serious concern with the recent emergence of Middle East respiratory syndrome and severe acute respiratory syndrome (SARS) in the human population. CoVs encode two large polyproteins, which are then processed into 15-16 nonstructural proteins (nsps) that make significant contributions to viral replication and transcription by assembling the RNA replicase complex. Among them, nsp9 plays an essential role in viral replication by forming a homodimer that binds single-stranded RNA. Thus, disrupting nsp9 dimerization is a potential anti-CoV therapy. However, different nsp9 dimer forms have been reported for alpha- and beta-CoVs, and no structural information is available for gamma-CoVs. Here we determined the crystal structure of nsp9 from the avian infectious bronchitis virus (IBV), a representative gamma-CoV that affects the economy of the poultry industry because it can infect domestic fowl. IBV nsp9 forms a homodimer via interactions across a hydrophobic interface, which consists of two parallel alpha helices near the carboxy terminus of the protein. The IBV nsp9 dimer resembles that of SARS-CoV nsp9, indicating that this type of dimerization is conserved among all CoVs. This makes disruption of the dimeric interface an excellent strategy for developing anti-CoV therapies. To facilitate this effort, we characterized the roles of six conserved residues on this interface using site-directed mutagenesis and a multitude of biochemical and biophysical methods. We found that three residues are critical for nsp9 dimerization and its abitlity to bind RNA.


Assuntos
Vírus da Bronquite Infecciosa/química , Multimerização Proteica , RNA Viral/química , Proteínas de Ligação a RNA/química , Proteínas Virais/química , Vírus da Bronquite Infecciosa/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Relação Estrutura-Atividade , Proteínas Virais/metabolismo
20.
Appl Biochem Biotechnol ; 183(1): 444-459, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28353041

RESUMO

Mesenchymal stem cells (MSCs) can migrate to the tumor site and integrate into the tumor tissue. As a delivery vehicle of antitumor factors, MSCs have been tried in various tumor therapies. NK4 can both inhibit the growth, metastasis, and invasion of tumor cells induced by hepatocyte growth factor (HGF) and suppress tumor angiogenesis that is independent on HGF/cellular-mesenchymal-to-transition factor pathway. Adenovirus can directly deliver NK4 for tumor treatment but may induce immunological rejection. We combined MSCs with an adenovirus vector to deliver NK4 for liver tumor treatment. This study detected the migration of MSCs to high metastasis liver carcinoma cells MHCC-97H in vitro, investigated the inhibitory effect of rAd-NK4-MSCs on the growth and metastasis of MHCC-97H cells, further explored the inhibitory mechanism of rAd-NK4-MSCs to MHCC-97H cell metastasis, and examined the inhibitory effect of rAd-NK4-MSCs on the migration of human umbilical vein endothelial cells (HUVECs) in vitro. In this study, migration experiment was used for the potential capacity of MSCs and inhibition on migration of rAd-NK4-MSCs. Western blot was used for detecting the inhibition mechanism of rAd-NK4-MSCs to MHCC-97H cells. And, animal transplantation experiment was used for the inhibition of rAd-NK4-MSCs in vivo. MSC migration assay showed MSCs can significantly migrate to MHCC-97H cells. Co-culture results indicated that rAd-NK4-MSCs significantly inhibited the proliferation and migration of MHCC-97H cells in vitro. Western blot results proved that rAd-NK4-MSCs inhibited MHCC-97H cell migration correlating with suppressing Erk1/2 phosphorylation. HUVEC migration experiment suggested that rAd-NK4-MSCs had a potential of inhibiting tumor angiogenesis. Animal transplantation experiment showed that the tumor growth was significantly inhibited in the rAd-NK4-MSC group. Taken together, this study proved that NK4-modified MSCs had an inhibitory effect on the growth and migration of MHCC-97H cells and tumor angiogenesis, which provided a new strategy for liver tumor target therapy.


Assuntos
Adenoviridae , Fator de Crescimento de Hepatócito , Neoplasias Hepáticas Experimentais/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Transdução Genética , Animais , Linhagem Celular Tumoral , Feminino , Fator de Crescimento de Hepatócito/biossíntese , Fator de Crescimento de Hepatócito/genética , Xenoenxertos , Humanos , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Ratos
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