RESUMO
Organ-specific cell-penetrating peptides (CPPs) are a class of molecules that can be highly effective at delivering therapeutic cargoes, and they are currently of great interest in cancer treatment strategies. Herein, we describe a new CPP (amino acid sequence serine-isoleucine-tyrosine-valine, or SIWV) that homes to glioblastoma multiforme (GBM) brain tumor tissues with remarkable specificity in vitro and in vivo. The SIWV sequence was identified from an isoform of annexin-A3 (AA3H), a membrane-interacting human protein. The mechanism of intracellular permeation is proposed to follow a caveolin-mediated endocytotic pathway, based on in vitro and in vivo receptor inhibition and genetic knockdown studies. Feasibility as a targeting agent for therapeutics is demonstrated in a GBM xenograft mouse model, where porous silicon nanoparticles (pSiNPs) containing the clinically relevant anticancer drug SN-38 are grafted with SIWV via a poly-(ethylene glycol) (PEG) linker. The formulation shows enhanced in vivo targeting ability relative to a formulation employing a scrambled control peptide, and significant (P < 0.05) therapeutic efficacy relative to free SN-38 in the GBM xenograft animal model.
Assuntos
Antineoplásicos/uso terapêutico , Peptídeos Penetradores de Células/química , Portadores de Fármacos/química , Glioblastoma/tratamento farmacológico , Irinotecano/uso terapêutico , Oligopeptídeos/química , Animais , Anexina A3/química , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Fragmentos de Peptídeos/química , Polietilenoglicóis/química , Silício/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We demonstrated that 19 out of 20 RNA residues in the guide region of crRNA can be replaced with DNA residues with high GC-contents. The cellular activity of the chimeric crRNAs to disrupt the target gene was comparable to that of the native crRNA.
Assuntos
Composição de Bases , Proteína 9 Associada à CRISPR/química , Sistemas CRISPR-Cas , DNA/química , RNA Guia de Cinetoplastídeos/química , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Clivagem do DNARESUMO
The present study was performed to characterize the differential molecular mechanisms of morphine and beta-endorphin which are injected intracerebroventiricularly in mice. In the immunoblot assay, the increases of phosphorylated extracellular signal-regulated protein kinase (pERK) as well as phosphorylated calcium/calmodulin-dependent protein kinase IIalpha (pCaMK-IIalpha) expression induced by noxious stimuli were attenuated by intracerebroventricular (i.c.v.) beta-endorphin pretreatment in the hypothalamus, but not by i.c.v. morphine pretreatment. In addition to these immunoblot results, immunohistochemical study also showed that the attenuation of pERK or pCaMK-IIalpha immunoreactivity elicited by i.c.v. pretreatment of beta-endorphin mainly occurred in the paraventricular nucleus of the hypothalamus (PVN). We also investigated the effect of morphine and beta-endorphin on pERK and pCaMK-IIalpha expression in the locus coeruleus (LC). I.c.v. injection of morphine significantly increased pERK as well as pCaMK-IIalpha expression in the locus coeruleus, while beta-endorphin increased only pCaMK-IIalpha in the LC. In addition, beta-endorphin significantly attenuated pERK expression induced by SP i.t. injection. These results suggest that the antinociceptive effects of supraspinally administered morphine and beta-endorphin are involved with differentially intracellular signal transduction molecules-pERK, pCaMK-IIalpha in the PVN and the LC.
Assuntos
Analgésicos Opioides/farmacologia , Química Encefálica/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Morfina/farmacologia , Dor/metabolismo , beta-Endorfina/farmacologia , Analgésicos Opioides/administração & dosagem , Animais , Western Blotting , Química Encefálica/efeitos dos fármacos , Formaldeído , Imuno-Histoquímica , Injeções Intraventriculares , Injeções Espinhais , Masculino , Camundongos , Camundongos Endogâmicos ICR , Morfina/administração & dosagem , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/fisiologia , Substância P , beta-Endorfina/administração & dosagemRESUMO
Although many studies which explore on the interaction between stress and antinociception have been conducted, most of them do not divide stress into emotional stress (ES) and physical stress (PS). In the present study, we investigated the differential effects of ES or PS on pain behaviors or on c-Fos immunoreactivity (IR) in the paraventricular nucleus (PVN) or arcuate nucleus (ArcN) using electrical footshock-witness model. In addition, alteration of pain behaviors or c-Fos IR following stress repetition was examined. The electrical foot shock was applied to PS animal group in one chamber, whereas the witness animal group in another chamber without any electrical foot shock was regarded as an ES. In each group, either single (10 stimuli /10 min/1 day) or repeated stress for 5 consecutive days was applied. Our results suggest that ES and PS appear to play differential roles in the regulation of nociception produced by various types of pain stimuli (formalin, substance P, glutamate or pro-inflammatory cytokines) and on c-Fos IR in the PVN or ArcN. Moreover, such antinociceptive effect or c-Fos IR appears to be modified following stress repetition.
Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Dor/psicologia , Núcleo Hipotalâmico Paraventricular/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Estresse Psicológico/metabolismo , Análise de Variância , Animais , Doença Crônica , Modelos Animais de Doenças , Ácido Glutâmico/administração & dosagem , Ácido Glutâmico/efeitos adversos , Imuno-Histoquímica , Injeções Espinhais , Interferon gama/administração & dosagem , Interferon gama/efeitos adversos , Interleucina-1beta/administração & dosagem , Interleucina-1beta/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Dor/induzido quimicamente , Dor/complicações , Dor/metabolismo , Limiar da Dor/fisiologia , Limiar da Dor/psicologia , Estatísticas não Paramétricas , Estresse Psicológico/complicações , Estresse Psicológico/psicologia , Substância P/administração & dosagem , Substância P/efeitos adversos , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/efeitos adversosRESUMO
The effect of repeated immobilization stress (RIS) on the expression of interleukin-1beta (IL-1beta) and types of cells that express IL-1beta in hippocampal CA1 region, striatum and paraventricular nucleus (PVN) were investigated in ICR mice. The RIS was induced daily for 2h for 4 consecutive days. In the immunohistochemical study, RIS increased IL-1beta immunoreactivities (IR) in the hippocampal CA1 region and striatum and PVN. The RIS also increased glial fibrillary acidic protein (GFAP) IR and complement receptor type 3 (OX-42) IR in the hippocampal CA1 regions and striatum but not PVN. In confocal immunofluorescence study, the IL-1beta IR increased by RIS were colocalized with only NeuN, but not GFAP or OX-42 in the hippocampal CA1 region, striatum and PVN. Our results indicate that RIS increases IL-1beta IR on neuron, but not astrocyte or microglia in the hippocampal CA1 region, striatum and PVN, suggesting that the IL-1beta IR on neuron may play an important role during RIS. In addition, GFAP and OX-42 increased by RIS may be involved indirectly in playing another role in the hippocampal CA1 region and striatum during RIS.
Assuntos
Interleucina-1beta/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Prosencéfalo/metabolismo , Estresse Psicológico/metabolismo , Regulação para Cima/fisiologia , Animais , Astrócitos/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Antígeno CD11b/análise , Antígeno CD11b/metabolismo , Doença Crônica , Corpo Estriado/metabolismo , Corpo Estriado/fisiopatologia , Proteína Glial Fibrilar Ácida/análise , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Imuno-Histoquímica , Interleucina-1beta/imunologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microglia/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Núcleo Hipotalâmico Paraventricular/fisiopatologia , Prosencéfalo/fisiopatologia , Restrição Física/psicologia , Estresse Psicológico/fisiopatologiaRESUMO
Several ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg3) are neuroprotective and antinociceptive agents. In this study, we assessed the effects of these ginsenosides following intracerebroventricular (i.c.v.) administration on the nociceptive behaviors induced by intrathecal injection of pro-inflammatory cytokines (tumor necrosis factor-a (TNF-alpha), interleukin-1 beta (IL-1 beta), and interferon-gamma (IFN-gamma)). The ginsenosides, Rb1, Rb2, Rc, Rd, Re, Rf and Rg1, significantly attenuated the nociceptive behavior induced by TNF-alpha, IL-1 beta, and IFN-gamma injection, but ginsenoside-Rg3 did not. These results suggest that several ginsenosides may regulate the nociceptive processing induced by pro-inflammatory cytokines.
Assuntos
Analgésicos/administração & dosagem , Comportamento Animal/efeitos dos fármacos , Citocinas/metabolismo , Ginsenosídeos/administração & dosagem , Mediadores da Inflamação/metabolismo , Dor/prevenção & controle , Animais , Citocinas/administração & dosagem , Modelos Animais de Doenças , Mediadores da Inflamação/administração & dosagem , Injeções Intraventriculares , Injeções Espinhais , Interferon gama/metabolismo , Interleucina-1beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Dor/metabolismo , Medição da Dor , Limiar da Dor/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC), are prevalent and debilitating health problems worldwide. Many types of drugs are used to treat IBDs, but they exhibit adverse effects such as vomiting, nausea, abdominal pain, diarrhea, etc. In order to overcome the limitations of current therapeutic drugs, scientists have searched for functional foods from natural resources. In this study, we investigated the anti-colitic effects of Wasabia japonica extract in a DSS-induced colitis model. Wasabi japonica is a plant of the Brassicaceae family that has recently been reported to exhibit properties of detoxification, anti-inflammation, and induction of apoptosis in cancer cells. In this study, we generated wasabi ethanol extract (WK) and assessed its anti-colitic effect. In addition, in order to improve delivery of the extract to the colon, WK was coated with 5% Eudragit S100 (WKE), after which the anti-colitic effects of WKE were assessed. In conclusion, WK prevented development of colitis through inhibition of the NF-kB signaling pathway and recovery of epithelial tight junctions. In addition, the anti-colitic effect of WK was enhanced by improving its delivery to the colon by coating the WK with Eudragit S100. Therefore, we suggest that wasabi can be used as a new functional food to prevent IBDs due to its anti-colitic effect.
Assuntos
Colite/prevenção & controle , Alimento Funcional/análise , NF-kappa B/imunologia , Extratos Vegetais/administração & dosagem , Wasabia/química , Animais , Colite/genética , Colite/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Transdução de Sinais/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/imunologiaRESUMO
Heat shock protein 90 (HSP90) stabilizing oncoproteins has been an attractive target in cancer therapy. 17-N-Allylamino-17-demethoxygeldanamycin (17-AAG), an HSP90 inhibitor, was tested in phase II/III clinical trials, but due to lack of efficacy, clinical evaluation of 17-AAG has achieved limited success, which led to resistance to 17-AAG. However, the mechanism of 17-AAG resistance has not clearly been identified. Here, we identified LGALS3BP (Lectin, galactoside-binding soluble 3 binding protein), a secretory glycoprotein, as a 17-AAG resistance factor. In the clinical reports, it was suggested that LGALS3BP was associated with low survival rate, development of cancer progression, and enhancement of metastasis in human cancers. As we confirmed that the LGALS3BP level was increased in 17-AAG-resistant cells (H1299_17R) compared with that of the parental cell line (H1299_17P), knockdown of LGALS3BP expression increased sensitivity to 17-AAG in H1299_17R cells. Overexpression of LGALS3BP also augmented PI3K/Akt and ERK signaling pathways. Furthermore, we determined that the PI3K/Akt signaling pathway was involved in LGALS3BP-mediated 17-AAG resistance in vitro and in vivo, demonstrating that LGALS3BP mediates the resistance against 17-AAG through PI3K/Akt activation rather than ERK activation. These findings suggest that LGALS3BP would be a target to overcome resistance to 17-AAG in lung cancer. For example, the combination of 17-AAG and PI3K/Akt inhibitor would effectively suppress acquired resistance to 17-AAG. In conclusion, targeting of LGALS3BP-mediated-specific survival signaling pathway in resistant cells may provide a novel therapeutic model for the cancer therapy. Mol Cancer Ther; 16(7); 1355-65. ©2017 AACR.
Assuntos
Antígenos de Neoplasias/genética , Benzoquinonas/administração & dosagem , Biomarcadores Tumorais/genética , Proteínas de Transporte/genética , Resistencia a Medicamentos Antineoplásicos/genética , Glicoproteínas/genética , Lactamas Macrocíclicas/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Benzoquinonas/efeitos adversos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Lactamas Macrocíclicas/efeitos adversos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
AIM OF THE STUDY: In this study, we examined the effect of different fractions and components of Chaga mushroom (Inonotus Obliquus) on viability and apoptosis of colon cancer cells. Among them, one component showed the most effective growth inhibition and was identified as ergosterol peroxide by NMR analysis. We investigated the anti-proliferative and apoptosis mechanisms of ergosterol peroxide associated with its anti-cancer activities in human colorectal cancer (CRC) cell lines and tested its anti-tumor effect on colitis-induced CRC developed by Azoxymethane (AOM)/Dextran sulfate sodium (DSS) in a mouse model. MATERIALS AND METHODS: We used MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, flow cytometry assays, Western blot analysis, colony formation assays, reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and AOM/DSS mouse models to study the molecular mechanism of metastatic activities in CRC cells. RESULTS: Ergosterol peroxide inhibited cell proliferation and also suppressed clonogenic colony formation in HCT116, HT-29, SW620 and DLD-1 CRC cell lines. The growth inhibition observed in these CRC cell lines was the result of apoptosis, which was confirmed by FACS analysis and Western blotting. Ergosterol peroxide inhibited the nuclear levels of ß-catenin, which ultimately resulted in reduced transcription of c-Myc, cyclin D1, and CDK-8. Ergosterol peroxide administration showed a tendency to suppress tumor growth in the colon of AOM/DSS-treated mice, and quantification of the IHC staining showed a dramatic decrease in the Ki67-positive staining and an increase in the TUNEL staining of colonic epithelial cells in AOM/DSS-treated mice by ergosterol peroxide for both prevention and therapy. CONCLUSION: Our data suggest that ergosterol peroxide suppresses the proliferation of CRC cell lines and effectively inhibits colitis-associated colon cancer in AOM/DSS-treated mice. Ergosterol peroxide down-regulated ß-catenin signaling, which exerted anti-proliferative and pro-apoptotic activities in CRC cells. These properties of ergosterol peroxide advocate its use as a supplement in colon cancer chemoprevention.