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1.
Theranostics ; 11(19): 9415-9430, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34646378

RESUMO

The feasibility of personalized medicine for cancer treatment is largely hampered by costly, labor-intensive and time-consuming models for drug discovery. Herein, establishing new pre-clinical models to tackle these issues for personalized medicine is urgently demanded. Methods: We established a three-dimensional tumor slice culture (3D-TSC) platform incorporating label-free techniques for time-course experiments to predict anti-cancer drug efficacy and validated the 3D-TSC model by multiphoton fluorescence microscopy, RNA sequence analysis, histochemical and histological analysis. Results: Using time-lapse imaging of the apoptotic reporter sensor C3 (C3), we performed cell-based high-throughput drug screening and shortlisted high-efficacy drugs to screen murine and human 3D-TSCs, which validate effective candidates within 7 days of surgery. Histological and RNA sequence analyses demonstrated that 3D-TSCs accurately preserved immune components of the original tumor, which enables the successful achievement of immune checkpoint blockade assays with antibodies against PD-1 and/or PD-L1. Label-free multiphoton fluorescence imaging revealed that 3D-TSCs exhibit lipofuscin autofluorescence features in the time-course monitoring of drug response and efficacy. Conclusion: This technology accelerates precision anti-cancer therapy by providing a cheap, fast, and easy platform for anti-cancer drug discovery.

2.
Pharmacol Res ; 173: 105870, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34500061

RESUMO

Colorectal cancer (CRC) is one of the most common causes of cancer-related death worldwide. Nannocystin ax (NAN), a 21-membered cyclodepsipeptide initially isolated from myxobacteria of the Nannocystis genus, was found to target the eukaryotic elongation factor 1A (eEF1A). The current study was designed to evaluate the anticancer effect and underlying mechanisms of NAN with in vitro and in vivo models. Results showed that NAN induced G1 phase cell cycle arrest and caspase-independent apoptosis in HCT116 and HT29 human CRC cells. NAN significantly downregulated cyclin D1 level in a short time, but NAN did not affect the transcription level and ubiquitin-dependent degradation of cyclin D1. Furthermore, NAN treatment directly targeted eEF1A and partially decreased the synthesis of new proteins, contributing to the downregulation of cyclin D1. Besides, NAN significantly suppressed tumor growth in the zebrafish xenograft model. In conclusion, NAN triggered G1 phase cell cycle arrest through cyclin D1 downregulation and eEF1A-targeted translation inhibition and promoted caspase-independent apoptosis in CRC cells.

3.
Comput Struct Biotechnol J ; 19: 2664-2675, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093983

RESUMO

Chromogranin A (CgA) is a hydrophilic glycoprotein released by post-ganglionic sympathetic neurons. CgA consists of a single peptide chain containing numerous paired basic residues, which are typical cleavage sites in prohormones to generate bioactive peptides. It is recognized as a diagnostic and prognostic serum marker for neuroendocrine tumours. Vasostatin-1 is one of the most conserved regions of CgA and has diverse inhibitory biological activities. In this study, a novel peptide fragment that contains three typical functional structures of Vasostatin-1 was synthesized. This unique bioengineered Vasostatin-1 Derived Peptide (named V1DP) includes a highly conserved domain between vertebrate species in its N-terminal region, comprising a disulphide bridge formed by two cysteine residues at amino acid positions 17 and 38, respectively. Besides, V1DP contains two significant tripeptide recognition sequences: the amino acid triplets, RGD and KGD. Our data demonstrated that V1DP could induce a dose-dependent relaxation of rat arterial smooth muscle and also increase the contraction activity of rat uterus smooth muscle. More importantly, we found that V1DP inhibits cancer cell proliferation, modulate the HUVEC cell migration, and exhibit anti-angiogenesis effect both in vitro and in vivo. We further investigated the actual mechanism of V1DP, and our results confirmed that V1DP involves inhibiting the vascular endothelial growth factor receptor (VEGFR) signalling. We docked V1DP to the apo structures of VEGFR2 and examined the stability of the peptide in the protein pockets. Our simulation and free energy calculations results indicated that V1DP can bind to the catalytic domain and regulatory domain pockets, depending on whether the conformational state of the protein is JM-in or JM-out. Taken together, our data suggested that V1DP plays a role as the regulator of endothelial cell function and smooth muscle pharmacological homeostasis. V1DP is a water-soluble and biologically stable peptide and could further develop as an anti-angiogenic drug for cancer treatment.

4.
Free Radic Biol Med ; 166: 104-115, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33600944

RESUMO

Glioblastoma (GBM) is one of the most prevalent malignant primary tumors in the human brain. Temozolomide (TMZ), the chemotherapeutic drug for GBM treatment, induces apoptosis. Unfortunately, apoptosis-resistance to TMZ results in treatment failure. GBM shows enhanced expression of NAD(P)H: quinone oxidoreductase 1 (NQO1). Recently, noptosis, a type of NQO1-dependent necrosis, was proposed. Here, we identified that tanshindiol B (TSB) inhibits GBM growth by induction of noptosis. TSB triggered significant cell death, which did not fit the criteria of apoptosis but oxidative stress-induced necrosis. Molecular docking, cellular thermal shift assay, and NQO1 activity assay revealed that TSB bind to and promptly activated NQO1 enzyme activity. As the substrate of NQO1, TSB induced oxidative stress, which resulted in dramatic DNA damage, poly (ADP-ribose) polymerase 1 (PARP1) hyperactivation, and NAD+ depletion, leading to necrotic cell death. These effects of TSB were completely abolished by specific NQO1 inhibitor dicoumarol (DIC). Furthermore, the c-Jun N-terminal kinase 1/2 (JNK1/2) plays an essential role in mediating TSB-induced cell death. Besides, TSB significantly suppressed tumor growth in a zebrafish xenograft model mediated by NQO1. In conclusion, these results showed that TSB was an NQO1 substrate and triggered noptosis of GBM. TSB exhibited anti-tumor potentials in GBM both in vitro and in vivo. This study provides a novel strategy for fighting GBM through the induction of noptosis.


Assuntos
Glioblastoma , Animais , Apoptose , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Simulação de Acoplamento Molecular , NAD(P)H Desidrogenase (Quinona)/genética , Necrose , Peixe-Zebra
5.
Methods Mol Biol ; 2218: 1-9, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606218

RESUMO

Zebrafish ovarian follicles are mainly composed of the oocyte and a thin layer of follicle cells. Recent studies have demonstrated extensive cell-cell interactions between the oocyte and surrounding follicle layer and that the two compartments communicate mostly through paracrine factors. To understand the paracrine communication within the follicle, it is essential to know the spatial expression patterns of genes in the two compartments. However, since the follicle layer is extremely thin and the oocytes are enormous in size in fish, it is often difficult to detect gene expression by traditional methods such as in situ hybridization. Separation of the oocyte and surrounding follicle layer followed by RT-PCR detection provides a sensitive way to reveal the expression of individual genes in the two compartments of the follicle. This chapter introduces a method for mechanic separation of the oocyte and follicle layer at full-grown stage for expression analysis. Since fish have similar follicle structure, this method may also be used in other species as well.


Assuntos
Expressão Gênica/fisiologia , Oócitos/fisiologia , Folículo Ovariano/fisiologia , Peixe-Zebra/fisiologia , Animais , Feminino , Ovário/fisiologia , Comunicação Parácrina/fisiologia
6.
Free Radic Biol Med ; 152: 336-347, 2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32234332

RESUMO

Glioblastoma (GBM) are the most malignant brain tumors in humans and have a very poor prognosis. Temozolomide (TMZ), the only chemotherapeutic drug for GBM treatment, induced apoptosis but frequently developed resistance. Non-apoptotic cell death offers an alternative strategy to fight cancers. Our previous studies showed that 2-methoxy-6-acetyl-7-methyljuglone (MAM), a natural product, induced necroptosis in lung and colon cancer cells. The current study is designed to investigate its therapeutic potentials for GBM with in vitro and in vivo models. The protein expression of NAD(P)H: quinone oxidoreductase 1 (NQO1) in human GBM specimens were detected by immunohistochemistry. Effect of MAM on NQO1 was measured by recombinant protein and cellular thermal shift assay. The roles of NQO1 activation, superoxide (O2-) generation, calcium (Ca2+) accumulation, and c-Jun N-terminal kinase (JNK1/2) activation in MAM-induced cell death in U87 and U251 glioma cells were investigated. The effect of MAM on tumor growth was tested with a U251 tumor xenograft zebrafish model. Results showed that the NQO1 expression is positively correlated with the degree of malignancy in GBM tissues. MAM could directly bind and activate NQO1. Furthermore, MAM treatment induced rapid O2- generation, cytosolic Ca2+ accumulation, and sustained JNK1/2 activation. In addition, MAM significantly suppressed tumor growth in the zebrafish model. In conclusion, MAM induced GBM cell death by triggering an O2-/Ca2+/JNK1/2 dependent programmed necrosis. NQO1 might be the potential target for MAM and mediated its anticancer effect. This non-apoptotic necrosis might have therapeutic potentials for GBM treatment.


Assuntos
Glioblastoma , Naftoquinonas , Animais , Apoptose , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , NAD , NAD(P)H Desidrogenase (Quinona)/genética , Naftoquinonas/farmacologia , Necrose , Peixe-Zebra
7.
Cell Rep ; 29(11): 3374-3384.e5, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31825822

RESUMO

During embryogenesis, various cell types emerge simultaneously from their common progenitors under the influence of intrinsic signals. Human embryonic stem cells can differentiate to diverse cell types of three embryonic lineages, making them an excellent system for understanding the regulatory mechanism that maintains the balance of different cell types in embryogenesis. In this report, we demonstrate that insulin-like growth factor (IGF) proteins are endogenously expressed during differentiation, and their temporal expression contributes to the cell fate diversity in mesoderm differentiation. Small molecule LY294002 inhibits the IGF pathway to promote cardiomyocyte differentiation while suppressing epicardial and noncardiac cell fates. LY294002-induced cardiomyocytes demonstrate characteristic cardiomyocyte features and provide insights into the molecular mechanisms underlying cardiac differentiation. We further show that LY294002 induces cardiomyocytes through CK2 pathway inhibition. This study elucidates the crucial roles of endogenous IGF in mesoderm differentiation and shows that the inhibition of the IGF pathway is an effective approach for generating cardiomyocytes.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Humanas/citologia , Transdução de Sinais , Somatomedinas/metabolismo , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Células Cultivadas , Cromonas/farmacologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mesoderma/citologia , Mesoderma/metabolismo , Morfolinas/farmacologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Inibidores de Proteínas Quinases/farmacologia
8.
Endocrinology ; 160(3): 568-586, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30668682

RESUMO

As a master hormone controlling growth and metabolism, GH is also known to regulate reproduction. Studies in mammals have shown that mutations in GH or its receptor (GHR) not only result in retardation in body growth but also reproductive dysfunctions in both sexes. However, the roles of GH in reproduction of other vertebrates are poorly defined. In this study, we created two zebrafish GH (gh1) mutant lines using CRISPR/Cas9. The mutant developed normally up to 14 days postfertilization (dpf); however, a high rate of mortality was observed afterward in both lines, and only a small number of mutant fish could survive to adult stage. The body growth of the mutants was significantly retarded in both sexes in a gene dose-dependent manner compared with their wild-type siblings. A severe dysfunction of gonadal development was observed in survived mutant females, with ovarian folliculogenesis being arrested completely at primary growth stage until 100 dpf. Interestingly, the folliculogenesis in the mutant resumed after months of delay with a certain number of follicles entering vitellogenic growth. As for male reproduction, although the spermatogenesis in mutant males seemed normal in adults, the GH-insufficient heterozygote showed an obvious delay of spermatogenesis (puberty onset) at early developmental stages. The adult mutant males could not breed with wild-type females through natural spawning; however, the sperm isolated from the mutant testes could fertilize eggs through artificial fertilization. This study provides further genetic evidence for the dependence of puberty onset on somatic growth, but not age, in fish.


Assuntos
Hormônio do Crescimento/fisiologia , Folículo Ovariano/crescimento & desenvolvimento , Espermatogênese , Peixe-Zebra/crescimento & desenvolvimento , Animais , Feminino , Masculino , Mutagênese Sítio-Dirigida , Peixe-Zebra/genética
9.
Oncotarget ; 9(62): 31958-31970, 2018 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-30174789

RESUMO

Angiogenesis is a hallmark for cancer development because it is essential for cancer growth and provides the route for cancer cell migration (metastasis). Understanding the mechanism of angiogenesis and developing drugs that target the process has therefore been a major focus for research on cancer therapy. In this study, we screened 114 FDA-approved anti-cancer drugs for their effects on angiogenesis in the zebrafish. Among those with positive effects, we chose to focus on Ponatinib (AP24534; Iclusig®) for further investigation. Ponatinib is an inhibitor of the tyrosine kinase BCR-ABL in chronic myeloid leukemia (CML), and its clinical trial has been approved by FDA for the treatment of the disease. In recent clinical trials, however, some side effects have been reported for Ponatinib, mostly on blood vessel disorders, raising the possibility that this drug may influence angiogenesis. In this study, we demonstrated that Ponatinib was able to suppress the formation of intersegmental vessels (ISV) and subintestinal vessels (SIV) in the zebrafish larvae. The anti-angiogenic effect of Ponatinib was further validated by other bioassays in human umbilical vein endothelial cells (HUVECs), including cell proliferation and migration, tube formation, and wound healing. Further experiments showed that Ponatinib inhibited VEGF-induced VEGFR2 phosphorylation and its downstream signaling pathways including Akt/eNOS/NO pathway and MAPK pathways (ERK and p38MAPK). Taken together, these results suggest that inhibition of VEGF signaling at its receptor level and downstream pathways may likely be responsible for the antiangiogenic activity of Ponatinib.

10.
Cell Death Dis ; 9(9): 879, 2018 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-30158533

RESUMO

Autophagy impairment is commonly implicated in the pathological characteristic of Alzheimer's disease (AD). Presenilin 1 (PS1) expression in human brain gradually decreases with age and its mutations account for the most common cases of early-onset familial Alzheimer's disease (FAD). The dominant autophagy phenotypes occur in PS1-knockout and PS1 mutant neurons; it is still unknown whether PS1 deficiency causes serious autophagy impairment in neural stem cells (NSCs). Herein, we generated the heterozygote and homozygote of PS1 knockout in human induced pluripotent stem cells (iPSCs) via CRISPR/Cas9-based gene editing and differentiated them into human NSCs. In these human PS1-deficient NSCs, reduced autophagosome formation and downregulated expression of autophagy-lysosome pathway (ALP)-related mRNAs, as well as proteins were observed. Mechanistically, ERK/CREB inhibition and GSK3ß activation had key roles in reducing TFEB expression in PS1-knockout NSCs. Pharmacological inhibition of GSK3ß upregulated the expression of TFEB and ALP-related proteins in PS1-knockout NSCs, whereas this effect could be blocked by CREB inhibition. These findings demonstrate that PS1 deficiency causes autophagy suppression in human NSCs via downregulating ERK/CREB signaling.


Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Autofagia/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Presenilina-1/deficiência , Autofagossomos/metabolismo , Autofagossomos/fisiologia , Encéfalo/metabolismo , Encéfalo/fisiologia , Sistemas CRISPR-Cas/fisiologia , Células Cultivadas , Regulação para Baixo/fisiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Lisossomos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Mutação/fisiologia , Células-Tronco Neurais/fisiologia , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia
11.
Int J Biol Sci ; 14(10): 1175-1185, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30123067

RESUMO

Cholesterol plays a key role in membrane protein function and signaling in endothelial cells. Thus, disturbing cholesterol trafficking is an effective approach for inhibiting angiogenesis. We recently identified astemizole (AST), an antihistamine drug, as a cholesterol trafficking inhibitor from a phenotypic screen. In this study, we found that AST induced cholesterol accumulation in the lysosome by binding to the sterol-sensing domain of Niemann-Pick disease, type C1 (NPC1), a lysosomal surface protein responsible for cholesterol transport. Inhibition of cholesterol trafficking by AST led to the depletion of membrane cholesterol, causing SREBP1 nuclear localization. The depletion of membrane cholesterol resulted in dissociation of mammalian target of rapamycin (mTOR) from the lysosomal surface and inactivation of mTOR signaling. These effects were effectively rescued by addition of exogenous cholesterol. AST inhibited endothelial cell proliferation, migration and tube formation in a cholesterol-dependent manner. Furthermore, AST inhibited zebrafish angiogenesis in a cholesterol-dependent manner. Together, our data suggest that AST is a new class of NPC1 antagonist that inhibits cholesterol trafficking in endothelial cells and angiogenesis.


Assuntos
Astemizol/uso terapêutico , Colesterol/metabolismo , Neovascularização Patológica/tratamento farmacológico , Serina-Treonina Quinases TOR/metabolismo , Células A549 , Transporte Biológico/efeitos dos fármacos , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Imunofluorescência , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteína C1 de Niemann-Pick/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética
12.
Mol Neurobiol ; 55(3): 2645-2652, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28421538

RESUMO

Nitric oxide (NO), a free radical gas, acts as a neurotransmitter or neuromodulator in the central nervous system (CNS). It has been widely explored as a mediator of neuroinflammation, neuronal damages, and neurodegeneration at its pathological levels. Recently, increasing evidence suggests that NO plays key roles in mediating adult neurogenesis, the process of neural stem cells (NSCs) to generate newborn neurons for replacing damaged neurons or maintaining the function of the brain. NO synthase (NOS) is a major enzyme catalyzing the generation of NO in the brain. Recent studies indicate that three homologous NOS isoforms are involved in the proliferation of NSCs and neurogenesis. Therefore, the impact of NOS isoforms on NSC functions needs to be elucidated. Here, we summarize the studies on the role of NO and NOS with different isoforms in NSC proliferation and neurogenesis with the focus on introducing action mechanisms involved in the regulation of NSC function. This growing research area provides the new insight into controlling NSC function via regulating NO microenvironment in the brain. It also provides the evidence on targeting NOS for the treatment of brain diseases.


Assuntos
Encéfalo/enzimologia , Neurogênese/fisiologia , Óxido Nítrico Sintase/fisiologia , Animais , Encéfalo/citologia , Humanos , Isoenzimas/fisiologia , Células-Tronco Neurais/enzimologia , Óxido Nítrico Sintase/química
13.
Cancer Lett ; 409: 91-103, 2017 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-28923401

RESUMO

Cholesterol is an important modulator of membrane protein function and signaling in endothelial cells, thus making it an emerging target for anti-angiogenic agents. In this study, we employed a phenotypic screen that detects intracellular cholesterol distribution in endothelial cells (HUVEC) and identified 13 existing drugs as cholesterol trafficking inhibitors. Cepharanthine, an approved drug for anti-inflammatory and cancer management use, was amongst the candidates, which was selected for in-depth mechanistic studies to link cholesterol trafficking and angiogenesis. Cepharanthine inhibited the endolysosomal trafficking of free-cholesterol and low-density lipoprotein in HUVEC by binding to Niemann-Pick disease, type C1 (NPC1) protein and increasing the lysosomal pH. The blockade of cholesterol trafficking led to a cholesterol-dependent dissociation of mTOR from the lysosomes and inhibition of its downstream signaling. Cepharanthine inhibited angiogenesis in HUVEC and in zebrafish in a cholesterol-dependent manner. Furthermore, cepharanthine suppressed tumor growth in vivo by inhibiting angiogenesis and it enhanced the antitumor activity of the standard chemotherapy cisplatin in lung and breast cancer xenografts in mice. Altogether, these results strongly support the idea that cholesterol trafficking is a viable drug target for anti-angiogenesis and that the inhibitors identified among existing drugs, such as cepharanthine, could be potential anti-angiogenic and antitumor agents.


Assuntos
Benzilisoquinolinas/farmacologia , Colesterol/metabolismo , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Animais , Animais Geneticamente Modificados , Antineoplásicos Fitogênicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzilisoquinolinas/administração & dosagem , Processos de Crescimento Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Sinergismo Farmacológico , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
14.
Cell Death Discov ; 3: 17051, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28845297

RESUMO

Hemorrhagic stroke occurs when a weakened vessel ruptures and bleeds into the surrounding brain, leading to high rates of death and disability worldwide. A series of complex pathophysiological cascades contribute to the risk of hemorrhagic stroke, and no therapies have proven effective to prevent hemorrhagic stroke. Stabilization of vascular integrity has been considered as a potential therapeutic target for hemorrhagic stroke. ROCKs, which belong to the serine/threonine protein kinase family and participate in the organization of actin cytoskeleton, have become attractive targets for the treatment of strokes. In this study, in vitro enzyme-based assays revealed that a new compound (FPND) with a novel scaffold identified by docking-based virtual screening could inhibit ROCK1 specifically at low micromolar concentration. Molecular modeling showed that FPND preferentially interacted with ROCK1, and the difference between the binding affinity of FPND toward ROCK1 and ROCK2 primarily resulted from non-polar contributions. Furthermore, FPND significantly prevented statin-induced cerebral hemorrhage in a zebrafish model. In addition, in vitro studies using the xCELLigence RTCA system, immunofluorescence and western blotting revealed that FPND prevented statin-induced cerebral hemorrhage by enhancing endothelial cell-cell junctions through inhibiting the ROCK-mediated VE-cadherin signaling pathway. As indicated by the extremely low toxicity of FPND against mice, it is safe and can potentially prevent vascular integrity loss-related diseases, such as hemorrhagic stroke.

15.
Curr Drug Targets ; 18(4): 455-462, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27033194

RESUMO

Rho-associated protein kinase (ROCK) is a serine-threonine kinase originally identified as a crucial regulator of actin cytoskeleton. Recent studies have defined new functions of ROCK as a critical component of diverse signaling pathways in neurons. In addition, inhibition of ROCK causes several biological events such as increase of neurite outgrowth, axonal regeneration, and activation of prosurvival Akt. Thus, it has attracted scientist's strong attentions and considered ROCK as a promising therapeutic target for the treatment of neurodegenerative disorders including Alzheimer disease, Parkinson's disease, Huntington';s disease, multiple sclerosis, and amyotrophic lateral sclerosis. However, ROCK has two highly homologous isoforms, ROCK1 and ROCK2. Accumulated evidences indicate that ROCK1 and ROCK2 might involve in distinct cellular functions in central nervous system (CNS) and neurodegenerative processes. This review summarizes recent updates regarding ROCK isoformspecific functions in CNS and the progress of ROCK inhibitors in preclinical studies for neurodegenerative diseases.


Assuntos
Doenças Neurodegenerativas/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Sistema Nervoso Central/metabolismo , Humanos , Terapia de Alvo Molecular , Doenças Neurodegenerativas/tratamento farmacológico , Transdução de Sinais
16.
Mol Biosyst ; 12(9): 2713-21, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27354305

RESUMO

Rho-associated protein kinase (ROCK) mediated the reorganization of the actin cytoskeleton and has been implicated in the spread and metastatic process of cancer. In this study, structure-based high-throughput virtual screening was used to identify candidate compounds targeting ROCK2 from a chemical library. Moreover, high-content screening based on neurite outgrowth of SH-SY5Y cells (a human neuroblastoma cell line) was used for accelerating the identification of compounds with characteristics of ROCK2 inhibitors. The effects of bioactive ROCK2 inhibitor candidates were further validated using other bioassays including cell migration and wound healing in SH-SY5Y cells. Through the combined virtual and high-content drug screening, the compound 1,3-benzodioxol-5-yl[1-(5-isoquinolinylmethyl)-3-piperidinyl]-methanone (BIPM) was identified as a novel and potent ROCK2 inhibitor. Exposure of SH-SY5Y cells to BIPM led to significant changes in neurite length, cell migration and actin stress fibers. Further experiments demonstrated that BIPM was able to significantly inhibit phosphorylation of cofilin, a regulatory protein of actin cytoskeleton. These results suggest that BIPM could be considered as a promising scaffold for the further development of ROCK2 inhibitors for anti-cancer metastasis.


Assuntos
Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/química , Quinases Associadas a rho/química , Sítios de Ligação , Biomarcadores , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Simulação por Computador , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Ativação Enzimática/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Fluxo de Trabalho , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
17.
Free Radic Biol Med ; 89: 1057-66, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26415025

RESUMO

Progressive degeneration and death of neurons are main causes of neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. Although some current medicines may temporarily improve their symptoms, no treatments can slow or halt the progression of neuronal death. In this study, a pyrimidine derivative, benzyl 7-(4-hydroxy-3-methoxyphenyl)-5-methyl-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (BHDPC), was found to attenuate dramatically the MPTP-induced death of dopaminergic neurons and improve behavior movement deficiency in zebrafish, supporting its potential neuroprotective activity in vivo. Further study in rat organotypic cerebellar cultures indicated that BHDPC was able to suppress MPP(+)-induced cell death of brain tissue slices ex vivo. The protective effect of BHDPC against MPP(+) toxicity was also effective in human neuroblastoma SH-SY5Y cells through restoring abnormal changes in mitochondrial membrane potential and numerous apoptotic regulators. Western blotting analysis indicated that BHDPC was able to activate PKA/CREB survival signaling and further up-regulate Bcl2 expression. However, BHDPC failed to suppress MPP(+)-induced cytotoxicity and the increase of caspase 3 activity in the presence of the PKA inhibitor H89. Taken together, these results suggest that BHDPC is a potential neuroprotectant with prosurvival effects in multiple models of neurodegenerative disease in vitro, ex vivo, and in vivo.


Assuntos
Apoptose/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Intoxicação por MPTP/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/toxicidade , Pirimidinas/farmacologia , Tetrazóis/farmacologia , 1-Metil-4-fenilpiridínio/toxicidade , Animais , Western Blotting , Células Cultivadas , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Herbicidas/toxicidade , Humanos , Locomoção/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Teóricos , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Rombencéfalo/efeitos dos fármacos , Rombencéfalo/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo
18.
Biochim Biophys Acta ; 1850(6): 1253-60, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25731980

RESUMO

BACKGROUND: Animal venoms contain a diverse array of proteins and enzymes that are toxic toward various physiological systems. However, there are also some practical medicinal uses for these toxins including use as anti-bacterial and anti-tumor agents. METHODS: In this study, we identified a nine-residue cryptic oligopeptide, KRFKKFFKK (EVP50) that is repeatedly encoded in tandem within vipericidin sequences. RESULTS: EVP50 displayed in vivo potent lethal toxicity to zebrafish larvae (LD50=6 µM) when the peptide's N-terminus was chemically conjugated to rhodamine B (RhoB). In vitro, RhoB-conjugated EVP50 (RhoB-EVP50) exhibited a concentration-dependent cytotoxic effect toward MCF-7 and MDA-MB-231 breast cancer cells. In MCF-7 cells, the RhoB-EVP50 nonapeptide accumulated inside the cells within minutes. In the cytoplasm, the RhoB-EVP50 induced extracellular calcium influx and intracellular calcium release. Membrane budding was also observed after incubation with micromolar concentrations of the fluorescent EVP50 conjugate. CONCLUSIONS: The conjugate's interference with calcium homeostasis, its intracellular accumulation and its induced membrane dysfunction (budding and vacuolization) seem to act in concert to disrupt the cell circuitry. Contrastively, unconjugated EVP50 peptide did not display neither toxic nor cytotoxic activities in our in vivo and in vitro models. GENERAL SIGNIFICANCE: The synergic mechanism of toxicity was restricted to the structurally modified encrypted vipericidin nonapeptide.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Catelicidinas/farmacologia , Oligopeptídeos/farmacologia , Rodaminas/farmacologia , Venenos de Víboras/química , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Neoplasias da Mama/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Catelicidinas/isolamento & purificação , Catelicidinas/metabolismo , Catelicidinas/toxicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/patologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Larva/efeitos dos fármacos , Dose Letal Mediana , Células MCF-7 , Dados de Sequência Molecular , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/metabolismo , Oligopeptídeos/toxicidade , Rodaminas/metabolismo , Rodaminas/toxicidade , Fatores de Tempo
19.
J Pharm Sci ; 103(11): 3576-3584, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25187229

RESUMO

This work addressed solubility and membrane permeability problems of Biopharmaceutics Classification System (BCS) Class IV glycoside scutellarin (SG) by developing a nanosuspension of its aglycone scutellarein (S) as a precursor. An S nanosuspension containing poloxamer 188 was prepared using antisolvent precipitation where hydroxypropyl-ß-cyclodextrin was utilized as a lyophilizing protectant. Particle size and polydispersity index after redispersion were 342.6 ± 18.2 and 0.32 ± 0.06 nm, respectively. The dissolution rate of the S nanosuspension was superior compared with the physical mixture. No free S, but SG and SG's isomer were detected in plasma following oral delivery of SG or S, S nanosuspension or physical mixture of S. The Cmax values of SG after dosing with the S nanosuspension were 12.0, 8.0, and 4.5-fold higher than the SG, S, or physical mixture, respectively. The Tmax and mean residence time (MRTlast ) of SG after dosing with the S nanosuspension were significantly shorter than S and SG. Treatments with SG, S, or S nanosuspensions reduced the hemorrhage rate in a zebrafish model, but the S nanosuspension exhibited the strongest rescue effect. This study highlights a new strategy to circumvent BCS Class IV flavonoid glycosides using a formulation of their aglycone as a precursor to accelerate oral absorption and improve bioactivity.


Assuntos
Apigenina/administração & dosagem , Glucuronatos/administração & dosagem , Nanoestruturas , Pró-Fármacos/administração & dosagem , 2-Hidroxipropil-beta-Ciclodextrina , Administração Oral , Animais , Apigenina/sangue , Apigenina/química , Apigenina/farmacocinética , Disponibilidade Biológica , Biotransformação , Hemorragia Cerebral/sangue , Hemorragia Cerebral/prevenção & controle , Química Farmacêutica , Modelos Animais de Doenças , Excipientes/química , Liofilização , Glucuronatos/sangue , Glucuronatos/química , Glucuronatos/farmacocinética , Nanotecnologia , Tamanho da Partícula , Poloxâmero/química , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Ratos Sprague-Dawley , Solubilidade , Tecnologia Farmacêutica/métodos , Peixe-Zebra , beta-Ciclodextrinas/química
20.
Free Radic Biol Med ; 74: 283-93, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24973649

RESUMO

Parkinson disease (PD) is a neurodegenerative disease with multifactorial etiopathogenesis. The discovery of drug candidates that act on new targets of PD is required to address the varied pathological aspects and modify the disease process. In this study, a small compound, 2-(5-methyl-1-benzofuran-3-yl)-N-(5-propylsulfanyl-1,3,4-thiadiazol-2-yl) acetamide (MBPTA) was identified as a novel Rho-associated protein kinase inhibitor with significant protective effects against 1-methyl-4-phenylpyridinium ion (MPP(+))-induced damage in SH-SY5Y neuroblastoma cells. Further investigation showed that pretreatment of SH-SY5Y cells with MBPTA significantly suppressed MPP(+)-induced cell death by restoring abnormal changes in nuclear morphology, mitochondrial membrane potential, and numerous apoptotic regulators. MBPTA was able to inhibit MPP(+)-induced reactive oxygen species (ROS)/NO generation, overexpression of inducible NO synthase, and activation of NF-κB, indicating the critical role of MBPTA in regulating ROS/NO-mediated cell death. Furthermore, MBPTA was shown to activate PI3K/Akt survival signaling, and its cytoprotective effect was abolished by PI3K and Akt inhibitors. The structural comparison of a series of MBPTA analogs revealed that the benzofuran moiety probably plays a crucial role in the anti-oxidative stress action. Taken together, these results suggest that MBPTA protects against MPP(+)-induced apoptosis in a neuronal cell line through inhibition of ROS/NO generation and activation of PI3K/Akt signaling.


Assuntos
Antioxidantes/farmacologia , Benzofuranos/farmacologia , Doença de Parkinson/tratamento farmacológico , Tiadiazóis/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Acetamidas/química , Ácidos Alcanossulfônicos/química , Antioxidantes/química , Benzofuranos/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citoproteção/efeitos dos fármacos , Descoberta de Drogas , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Terapia de Alvo Molecular , Proteína Oncogênica v-akt/metabolismo , Oxidantes/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Compostos de Piridínio/toxicidade , Transdução de Sinais/efeitos dos fármacos , Tiadiazóis/química
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