RESUMO
BACKGROUND & AIMS: Mitochondrial dysfunction is considered a pathogenic linker in the development of non-alcoholic steatohepatitis (NASH). Inappropriate mitochondrial protein-quality control, possibly induced by insufficiency of the mitochondrial matrix caseinolytic protease P (ClpP), can potentially cause mitochondrial dysfunction. Herein, we aimed to investigate hepatic ClpP levels in a diet-induced model of NASH and determine whether supplementation of ClpP can ameliorate diet-induced NASH. METHODS: NASH was induced by a high-fat/high-fructose (HF/HFr) diet in C57BL/6J mice. Stress/inflammatory signals were induced in mouse primary hepatocytes (MPHs) by treatment with palmitate/oleate (PA/OA). ClpP levels in hepatocytes were reduced using the RNAi-mediated gene knockdown technique but increased through the viral transduction of ClpP. ClpP activation was induced by administering a chemical activator of ClpP. RESULTS: Hepatic ClpP protein levels in C57BL/6J mice fed a HF/HFr diet were lower than the levels in those fed a normal chow diet. PA/OA treatment also decreased the ClpP protein levels in MPHs. Overexpression or activation of ClpP reversed PA/OA-induced mitochondrial dysfunction and stress/inflammatory signal activation in MPHs, whereas ClpP knockdown induced mitochondrial dysfunction and stress/inflammatory signals in these cells. On the other hand, ClpP overexpression or activation improved HF/HFr-induced NASH characteristics such as hepatic steatosis, inflammation, fibrosis, and injury in the C57BL/6J mice, whereas ClpP knockdown further augmented steatohepatitis in mice fed a HF/HFr diet. CONCLUSIONS: Reduced ClpP expression and subsequent mitochondrial dysfunction are key to the development of diet-induced NASH. ClpP supplementation through viral transduction or chemical activation represents a potential therapeutic strategy to prevent diet-induced NASH. LAY SUMMARY: Western diets, containing high fat and high fructose, often induce non-alcoholic steatohepatitis (NASH). Mitochondrial dysfunction is considered pathogenically linked to diet-induced NASH. We observed that the mitochondrial protease ClpP decreased in the livers of mice fed a western diet and supplementation of ClpP ameliorated western diet-induced NASH.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Dieta Hiperlipídica/efeitos adversos , Suplementos Nutricionais , Modelos Animais de Doenças , Endopeptidase Clp , Frutose/efeitos adversos , Frutose/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Ácido Oleico/metabolismo , Peptídeo Hidrolases/metabolismoRESUMO
BACKGROUND AND AIMS: Bile acids (BAs) are important regulators of metabolism and energy balance, but excess BAs cause cholestatic liver injury. The histone methyltransferase mixed-lineage leukemia-4 (MLL4) is a transcriptional coactivator of the BA-sensing nuclear receptor farnesoid X receptor (FXR) and epigenetically up-regulates FXR targets important for the regulation of BA levels, small heterodimer partner (SHP), and bile salt export pump (BSEP). MLL4 expression is aberrantly down-regulated and BA homeostasis is disrupted in cholestatic mice, but the underlying mechanisms are unclear. APPROACH AND RESULTS: We examined whether elevated microRNA-210 (miR-210) in cholestatic liver promotes BA-induced pathology by inhibiting MLL4 expression. miR-210 was the most highly elevated miR in hepatic SHP-down-regulated mice with elevated hepatic BA levels. MLL4 was identified as a direct target of miR-210, and overexpression of miR-210 inhibited MLL4 and, subsequently, BSEP and SHP expression, resulting in defective BA metabolism and hepatotoxicity with inflammation. miR-210 levels were elevated in cholestatic mouse models, and in vivo silencing of miR-210 ameliorated BA-induced liver pathology and decreased hydrophobic BA levels in an MLL4-dependent manner. In gene expression studies, SHP inhibited miR-210 expression by repressing a transcriptional activator, Kruppel-like factor-4 (KLF4). In patients with primary biliary cholangitis/cirrhosis (PBC), hepatic levels of miR-210 and KLF4 were highly elevated, whereas nuclear levels of SHP and MLL4 were reduced. CONCLUSIONS: Hepatic miR-210 is physiologically regulated by SHP but elevated in cholestatic mice and patients with PBC, promoting BA-induced liver injury in part by targeting MLL4. The miR-210-MLL4 axis is a potential target for the treatment of BA-associated hepatobiliary disease.
Assuntos
Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Ácidos e Sais Biliares , Colestase/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Cirrose Hepática Biliar/metabolismo , MicroRNAs/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Ácidos e Sais Biliares/biossíntese , Ácidos e Sais Biliares/metabolismo , Colestase/complicações , Perfilação da Expressão Gênica , Humanos , Fator 4 Semelhante a Kruppel , Cirrose Hepática Biliar/etiologia , Camundongos , Ativação TranscricionalRESUMO
The bile acid (BA) nuclear receptor, farnesoid X receptor (FXR/NR1H4), maintains metabolic homeostasis by transcriptional control of numerous genes, including an intestinal hormone, fibroblast growth factor-19 (FGF19; FGF15 in mice). Besides activation by BAs, the gene-regulatory function of FXR is also modulated by hormone or nutrient signaling-induced post-translational modifications. Recently, phosphorylation at Tyr-67 by the FGF15/19 signaling-activated nonreceptor tyrosine kinase Src was shown to be important for FXR function in BA homeostasis. Here, we examined the role of this FXR phosphorylation in cholesterol regulation. In both hepatic FXR-knockout and FXR-knockdown mice, reconstitution of FXR expression up-regulated cholesterol transport genes for its biliary excretion, including scavenger receptor class B member 1 (Scarb1) and ABC subfamily G member 8 (Abcg5/8), decreased hepatic and plasma cholesterol levels, and increased biliary and fecal cholesterol levels. Of note, these sterol-lowering effects were blunted by substitution of Phe for Tyr-67 in FXR. Moreover, consistent with Src's role in phosphorylating FXR, Src knockdown impaired cholesterol regulation in mice. In hypercholesterolemic apolipoprotein E-deficient mice, expression of FXR, but not Y67F-FXR, ameliorated atherosclerosis, whereas Src down-regulation exacerbated it. Feeding or treatment with an FXR agonist induced Abcg5/8 and Scarb1 expression in WT, but not FGF15-knockout, mice. Furthermore, FGF19 treatment increased occupancy of FXR at Abcg5/8 and Scarb1, expression of these genes, and cholesterol efflux from hepatocytes. These FGF19-mediated effects were blunted by the Y67F-FXR substitution or Src down-regulation or inhibition. We conclude that phosphorylation of hepatic FXR by FGF15/19-induced Src maintains cholesterol homeostasis and protects against atherosclerosis.
Assuntos
Colesterol/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Hepatócitos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Quinases da Família src/metabolismo , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Ácidos e Sais Biliares/metabolismo , Colesterol/sangue , Regulação para Baixo , Fatores de Crescimento de Fibroblastos/deficiência , Fatores de Crescimento de Fibroblastos/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Transdução de Sinais , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
BACKGROUND The aim of this research was to compare the effects of contrast bath therapy (CBT) and contrast therapy (CT) using infrared (IR) and cryotherapy (CR) on blood flow, muscle tone, and pain in the forearm. MATERIAL AND METHODS Twenty healthy individuals participated in this study. Each participant received 2 kinds of CT separated by a week. CBT involved immersion in hot water (38-40°C) for 4 minutes, followed by 1 minute of immersion in cold water (12-14°C) for four rotations. CT using IR and CR was performed in the same manner as CBT. RESULTS The variables measured were blood flow, muscle tone, and pain before and after intervention. Both types of CT produced fluctuations in the blood flow (P<0.05). The pain threshold increased on both therapies; a significant increase was noted with IR and CR (P<0.05) therapies. Muscle elasticity was induced and stiffness was reduced with all therapies (P<0.05). IR and CR resulted in significant changes (P<0.05) in blood flow as compared with the CBT. CONCLUSIONS The results of this study suggest that CT using IR and CR is more effective in improving blood flow than CBT and has the same effect on muscle tone and pain. Nonetheless, using IR and CR is efficient with regard to mobility and maintaining temperature; therefore, it would be convenient to use these in clinical settings. Further studies involving CT should be carried out to determine whether our findings are clinically relevant.
Assuntos
Crioterapia/métodos , Temperatura Alta/uso terapêutico , Hidroterapia/métodos , Raios Infravermelhos/uso terapêutico , Tono Muscular/fisiologia , Limiar da Dor/fisiologia , Fluxo Sanguíneo Regional/fisiologia , Adulto , Elasticidade/fisiologia , Feminino , Antebraço/fisiologia , Voluntários Saudáveis , Humanos , Fluxometria por Laser-Doppler , Masculino , Músculo Esquelético/fisiologia , Manejo da Dor/métodos , Temperatura Cutânea/fisiologia , Temperatura Cutânea/efeitos da radiação , Adulto JovemRESUMO
Ferritin cage nanoparticles are promising platforms for targeted delivery of imaging and therapeutic agents because their cage structure can accommodate small molecules and their surfaces can be decorated with multiple functionalities. However, selective targeting is still a challenge for translating ferritin-based nanomedicines into the clinic, especially for heterogeneous diseases such as cancer. Targeting peptides can be genetically fused onto the surface of a ferritin cage, forming peptide bunches on nanocages (PBNCs) that offer synergistic increases in binding avidity. Here, we utilized two sites of the ferritin monomer, the N-terminus and the loop between the fourth and fifth helices, which are exposed on the surface of the assembled 24-subunit ferritin cage, to ligate one or two types of peptides to achieve "super affinity" and bispecificity, respectively. PBNCs formed by ligation of the IL-4 receptor-targeting peptide, AP1, to both sites (48AP1-PBNCs) tethered IL-4R, expressing tumor cells with greater affinity than did PBNCs with AP1 ligated to a single site (24AP1-PBNCs). Moreover, bispecific PBNCs containing 24 RGD peptides and 24 AP1 peptides (24RGD/24AP1-PBNCs) were capable of independently targeting cells expressing the corresponding receptors. Bispecific and superaffinity PBNCs could be useful for efficient targeting of ferritin-based therapeutic/diagnostic agents in a clinical setting.
Assuntos
Ferritinas/química , Nanopartículas Metálicas/química , Oligopeptídeos/química , Linhagem Celular Tumoral , Humanos , Ligantes , Oligopeptídeos/metabolismo , Ligação Proteica , Receptores de Interleucina-4/metabolismoRESUMO
OBJECTIVE: Obesity-associated nonalcoholic fatty liver disease (NAFLD) is a leading cause of liver failure and death. However, the pathogenesis of NAFLD and its severe form, nonalcoholic steatohepatitis (NASH), is poorly understood. The energy sensor, AMP-activated protein kinase (AMPK), has decreased activity in obesity and NAFLD, but the mechanisms are unclear. Here, we examined whether obesity-induced miR-802 has a role in promoting NASH by targeting AMPK. We also investigated whether miR-802 and AMPK have roles in modulating beneficial therapeutic effects mediated by obeticholic acid (OCA), a promising clinical agent for NASH. METHODS: Immunoblotting, luciferase assays, and RNA-protein interaction studies were performed to test whether miR-802 directly targets AMPK. The roles of miR-802 and AMPK in NASH were examined in mice fed a NASH-promoting diet. RESULTS: Hepatic miR-802 and AMPK levels were inversely correlated in both NAFLD patients and obese mice. MicroRNA in silico analysis, together with biochemical studies in hepatic cells, suggested that miR-802 inhibits hepatic expression of AMPK by binding to the 3' untranslated regions of both human AMPKα1 and mouse Ampkß1. In diet-induced NASH mice, OCA treatment reduced hepatic miR-802 levels and improved AMPK activity, ameliorating steatosis, inflammation, and apoptosis, but these OCA-mediated beneficial effects on NASH pathologies, particularly reducing apoptosis, were reversed by overexpression of miR-802 or downregulation of AMPK. CONCLUSIONS: These results indicate that miR-802 inhibits AMPK by directly targeting Ampkß1, promoting NAFLD/NASH in mice. The miR-802-AMPK axis that modulates OCA-mediated beneficial effects on NASH may represent a new therapeutic target.
Assuntos
MicroRNAs , Hepatopatia Gordurosa não Alcoólica , Camundongos , Humanos , Animais , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteínas Quinases Ativadas por AMP , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Camundongos Obesos , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
Activation of farnesoid X receptor (FXR) by obeticholic acid (OCA) reduces hepatic inflammation and fibrosis in patients with primary biliary cholangitis (PBC), a life-threatening cholestatic liver failure. Inhibition of bromodomain-containing protein 4 (BRD4) also has antiinflammatory, antifibrotic effects in mice. We determined the role of BRD4 in FXR function in bile acid (BA) regulation and examined whether the known beneficial effects of OCA are enhanced by inhibiting BRD4 in cholestatic mice. Liver-specific downregulation of BRD4 disrupted BA homeostasis in mice, and FXR-mediated regulation of BA-related genes, including small heterodimer partner and cholesterol 7 alpha-hydroxylase, was BRD4 dependent. In cholestatic mice, JQ1 or OCA treatment ameliorated hepatotoxicity, inflammation, and fibrosis, but surprisingly, was antagonistic in combination. Mechanistically, OCA increased binding of FXR, and the corepressor silencing mediator of retinoid and thyroid hormone receptor (SMRT) decreased NF-κB binding at inflammatory genes and repressed the genes in a BRD4-dependent manner. In patients with PBC, hepatic expression of FXR and BRD4 was significantly reduced. In conclusion, BRD4 is a potentially novel cofactor of FXR for maintaining BA homeostasis and hepatoprotection. Although BRD4 promotes hepatic inflammation and fibrosis in cholestasis, paradoxically, BRD4 is required for the antiinflammatory, antifibrotic actions of OCA-activated FXR. Cotreatment with OCA and JQ1, individually beneficial, may be antagonistic in treatment of liver disease patients with inflammation and fibrosis complications.
Assuntos
Colestase/tratamento farmacológico , Colestase/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/antagonistas & inibidores , Animais , Azepinas/administração & dosagem , Azepinas/farmacologia , Ácidos e Sais Biliares/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ácido Quenodesoxicólico/administração & dosagem , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/farmacologia , Colestase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , Modelos Animais de Doenças , Interações Medicamentosas , Técnicas de Silenciamento de Genes , Humanos , Fígado/metabolismo , Cirrose Hepática Biliar/tratamento farmacológico , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Correpressor 2 de Receptor Nuclear/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triazóis/administração & dosagem , Triazóis/farmacologiaRESUMO
The lack of molecular targets and targeting probes remains a major drawback for targeted imaging and drug delivery in lung cancer. In this study, we exploited in vivo phage display to identify a novel targeting probe that homes to the tumor in a K-rasLA2 mutant mouse lung cancer model. Compared with other candidate peptides selected from 5 rounds of phage display, the CRQTKN peptide homed to tumor nodules in the lung of mutant mice at higher levels. Photoacoustic tomography of mutant mice detected lung tumors via tumor homing of the near-infrared fluorescence dye-labeled CRQTKN peptide. Ex vivo photoacoustic images of isolated organs further demonstrated tumor homing of the CRQTKN peptide, whereas minimal accumulation was observed in control organs, such as the liver. Compared with untargeted liposomes and doxorubicin, doxorubicin-loaded liposomes whose surface was modified with the CRQTKN peptide more efficiently delivered doxorubicin and reduced the number or size of tumor lesions in K-rasLA2 mutant mice. Analysis of hematologic parameters and liver and kidney function showed no significant systemic side effects by the treatments. Affinity-based identification was used to detect TNF receptor superfamily member 19L (TNFRSF19L), which was upregulated in lung tumors of mutant mice, as the receptor for the CRQTKN peptide. In conclusion, these results suggest that the CRQTKN peptide is a promising targeting probe for photoacoustic-guided detection and drug delivery to lung cancer, and acts by binding to TNFRSF19L. SIGNIFICANCE: These findings present a new tumor-targeting probe for photoacoustic-guided detection and drug delivery.
Assuntos
Neoplasias Pulmonares/diagnóstico por imagem , Peptídeos/administração & dosagem , Técnicas Fotoacústicas/métodos , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos , Humanos , Lipossomos/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Biblioteca de Peptídeos , Peptídeos/farmacocinética , Receptores do Fator de Necrose Tumoral/análise , Receptores do Fator de Necrose Tumoral/metabolismoRESUMO
Transforming growth factor-beta1 (TGF-beta1) is an important mediator of tissue fibrosis, including liver cirrhosis. Ribbon-type antisense oligonucleotide to TGF-beta1 (TGF-beta1 RiAS) was designed and combined with cationic peptide derived from the nuclear localization signal of human immunodeficiency virus-1 Tat protein for enhanced cellular uptake. When Hepa1c1c7 cells were transfected with TGF-beta1 RiAS, the level of TGF-beta1 mRNA was reduced by >70%. TGF-beta1 RiAS, mismatched RiAS, and normal saline were each injected into mice via the tail vein, beginning the week after intraperitoneal CCl4 injection and continuing for 7 weeks, in order to determine whether TGF-beta1 RiAS prevents the fibrotic changes induced by the CCl4 injection. After 8 weeks of the experiment, all of the mice treated with TGF-beta1 RiAS survived, compared to 50% of the control group and 65% of the mismatched RiAS-treated group. Upon examining the biochemical effects on the liver, TGF-beta1 mRNA levels were reduced significantly only in the TGF-beta1 RiAS-treated group. Immunohistochemical studies showed a reduced accumulation of collagen and alpha-smooth muscle actin. Our experimental results suggest that ribbon antisense to TGF-beta1, with efficient uptake, effectively blocks the expression of TGF-beta1 and prevents fibrosis of the liver.
Assuntos
Cirrose Hepática/prevenção & controle , Conformação de Ácido Nucleico , RNA Antissenso/química , RNA Antissenso/farmacologia , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Animais , Sequência de Bases , Tetracloreto de Carbono , Colágeno/biossíntese , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Peptídeos/metabolismo , RNA Antissenso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida , Transfecção , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Various non-viral delivery systems for small interfering RNAs (siRNA) have been developed. Such delivery systems generally exhibit tightly formed spherical structures. While such carriers have demonstrated good transfection activity in mono-layered cell systems, effects against solid tumors are often less apparent and difficult to demonstrate, likely due to the rigid structures of the carriers, which may prevent penetration to deeper regions within tumor tissue. Herein, we developed a flexible nanocarrier (FNC) system that is able to penetrate to deeper regions within tumor tissue. Specifically, we employed previously found flexible polyplexes comprised of siRNA and poly-l-lysine as wick structures for the preparation of FNCs. FNCs were constructed by coating the wick structures with lipids using a liposomal membrane fusion method. The diameters of the resulting FNCs were ca. 170nm, and the shapes were non-spherical. Lipid coating was confirmed using a nuclease resistance assay. Furthermore, FNCs showed significant RNA interference effects, comparable to Lipofectamine 2000, in a mono-layered cell system. To accelerate tumor penetration, the FNC surface was modified with polyethylene glycol (PEG) and the tight junction opener peptide AT1002. Surface-modified FNCs demonstrated effective penetrability into a cancer spheroid. Thus, we developed a novel and unique tumor-penetrable siRNA FNC system.
Assuntos
Técnicas de Transferência de Genes , Nanopartículas , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Animais , Linhagem Celular Tumoral , Lipídeos/química , Camundongos , Neoplasias/patologia , Tamanho da Partícula , Polietilenoglicóis/química , RNA Interferente Pequeno/farmacocinética , Distribuição Tecidual , Transfecção/métodosRESUMO
Cellular cross-talk between tumors and M2-polarized tumor-associated macrophages (TAM) favors tumor progression. Upregulation of IL4 receptor (IL4R) is observed in diverse tumors and TAMs. We tested whether an IL4R-targeted proapoptotic peptide could inhibit tumor progression. The IL4R-binding peptide (IL4RPep-1) preferentially bound to IL4R-expressing tumor cells and M2-polarized macrophages both in vitro and in 4T1 breast tumors in vivo To selectively kill IL4R-expressing cells, we designed an IL4R-targeted proapoptotic peptide, IL4RPep-1-K, by adding the proapoptotic peptide (KLAKLAK)2 to the end of IL4RPep-1. IL4RPep-1-K exerted selective cytotoxicity against diverse IL4R-expressing tumor cells and M2-polarized macrophages. Systemic administration of IL4RPep-1-K inhibited tumor growth and metastasis in 4T1 breast tumor-bearing mice. Interestingly, IL4RPep-1-K treatment increased the number of activated cytotoxic CD8+ T cells while reducing the numbers of immunosuppressive regulatory T cells and M2-polarized TAMs. No significant systemic side effects were observed. These results suggest that IL4R-targeted proapoptotic peptide has potential for treating diverse IL4R-expressing cancers. Mol Cancer Ther; 16(12); 2803-16. ©2017 AACR.
Assuntos
Peptídeos/metabolismo , Receptores de Interleucina-4/metabolismo , Animais , Apoptose , Feminino , Humanos , Camundongos , Metástase Neoplásica , Transdução de SinaisRESUMO
The overall prognosis of conventional chemotherapy for the treatment of bladder cancer is poor and reduction of its systemic side effects remains an unsolved issue. Targeted therapy for bladder cancer could improve therapeutic efficacy and reduce side effects. This study investigated a hybrid peptide (named Bld-1-KLA) composed of the CSNRDARRC peptide (Bld-1), which binds to bladder tumor cells, and the d-KLAKLAKKLAKLAK (KLA) peptide, which disrupts mitochondrial membrane and induces apoptotic cell death, as a bladder cancer-targeted therapeutic agent. Bld-1-KLA selectively bound to HT1376 bladder tumor cells and efficiently internalized into the cells but not to other types of tumor and normal cell lines. Bld-1-KLA exerted cytotoxic effects selectively to HT1376 cells (LC50=41.5µM), but not to other types of cells. Pretreatment of cells with Bld-1 inhibited the binding and cytotoxicity by Bld-1-KLA in HT1376 cells. It induced apoptosis of bladder tumor cells, while Bld-1 or KLA alone showed much lesser effect on apoptosis, and was co-localized in mitochondria. Bld-1-KLA was stable up to 24h in serum. In vivo fluorescence imaging showed that homing of Bld-1-KLA in the tumor in HT1376 tumor-bearing nude mice was greater than that of the control peptide-KLA after intravenous injection. Treatment of tumor-bearing mice with Bld-1-KLA, compared to the control peptide-KLA, induced apoptosis of tumor cells and inhibited tumor growth more efficiently. No significant side effects on body weight and the liver and myeloid function were observed in mice treated with Bld-1-KLA. These results suggest that Bld-1-KLA is a promising therapeutic agent for targeted therapy of bladder cancer.
Assuntos
Antineoplásicos/administração & dosagem , Peptídeos/administração & dosagem , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Peptídeos/farmacocinética , Peptídeos/uso terapêutico , Carga Tumoral/efeitos dos fármacos , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologiaRESUMO
Transforming growth factor-beta1 (TGF-beta1) is an important mediator of glomerulosclerosis and tubulointerstitial fibrosis in renal diseases. We designed ribbon-type antisense oligos of TGF-beta1, TGF-beta1 RiAS, and combined them with a short peptide of the nuclear localization signal to form a transfection complex of DNA/peptide/liposomes (DPL) for enhanced cellular uptake. When H4IIE cells were transfected with TGF-beta1 RiAS, the level of TGF-beta1 mRNA was reduced by >70%. We then examined the ratio of the kidney weight per body weight in rats. Whereas the weight ratio was 0.47% for the normal kidney, the ratio was 0.99% on day 5 after unilateral ureteric obstruction (UUO). The ratios were 0.95% with PBS injection, 1.07% with scrambled RiAS, and 0.68% with TGF-beta1 RiAS. When examined for TGF-beta1 expression in the tissue, the level of TGF-beta1 mRNA was also significantly reduced following treatment with TGF-beta1 RiAS. Further, physical changes such as diminished dilation, atrophy, as well as apoptosis caused by UUO were also found to be markedly reduced by TGF-beta1 RiAS. The results show that ribbon antisense to TGF-beta1 when combined with efficient uptake can effectively block TGF-beta1 expression and preserve tissue integrity in kidneys with UUO.
Assuntos
DNA Antissenso/genética , DNA Antissenso/metabolismo , Rim/metabolismo , Rim/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Apoptose , Linhagem Celular , Humanos , Imuno-Histoquímica , Rim/lesões , Lipossomos/metabolismo , Masculino , Conformação de Ácido Nucleico , Tamanho do Órgão/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1RESUMO
A growing body of evidence suggests that pathological lesions express tissue-specific molecular targets or biomarkers within the tissue. Interleukin-4 receptor (IL-4R) is overexpressed in many types of cancer cells, including lung cancer. Here we investigated the properties of IL-4R-binding peptide-1 (IL4RPep-1), a CRKRLDRNC peptide, and its ability to target the delivery of liposomes to lung tumor. IL4RPep-1 preferentially bound to H226 lung tumor cells which express higher levers of IL-4R compared to H460 lung tumor cells which express less IL-4R. Mutational analysis revealed that C1, R2, and R4 residues of IL4RPep-1 were the key binding determinants. IL4RPep-1-labeled liposomes containing doxorubicin were more efficiently internalized in H226 cells and effectively delivered doxorubicin into the cells compared to unlabeled liposomes. In vivo fluorescence imaging of nude mice subcutaneously xenotransplanted with H226 tumor cells indicated that IL4RPep-1-labeled liposomes accumulate more efficiently in the tumor and inhibit tumor growth more effectively compared to unlabeled liposomes. Interestingly, expression of IL-4R was high in vascular endothelial cells of tumor, while little was detected in vascular endothelial cells of control organs including the liver. IL-4R expression in cultured human vascular endothelial cells was also up-regulated when activated by a pro-inflammatory cytokine tumor necrosis factor-α. Moreover, the up-regulation of IL-4R expression was observed in primary human lung cancer tissues. These results indicate that IL-4R-targeting nanocarriers may be a useful strategy to enhance drug delivery through the recognition of IL-4R in both tumor cells and tumor endothelial cells.
Assuntos
Neoplasias Pulmonares/metabolismo , Oligopeptídeos/administração & dosagem , Receptores de Interleucina-4/metabolismo , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Doxorrubicina/uso terapêutico , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lipossomos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos Nus , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêuticoRESUMO
Early decision on tumor response after anti-cancer treatment is still an unmet medical need. Here we investigated whether in vivo imaging of apoptosis using linear and cyclic (disulfide-bonded) form of ApoPep-1, a peptide that recognizes histone H1 exposed on apoptotic cells, at an early stage after treatment could predict tumor response to the treatment later. Treatment of stomach tumor cells with cistplatin or cetuximab alone induced apoptosis, while combination of cisplatin plus cetuximab more efficiently induced apoptosis, as detected by binding with linear and cyclic form of ApoPep-1. However, the differences between the single agent and combination treatment were more remarkable as detected with the cyclic form compared to the linear form. In tumor-bearing mice, apoptosis imaging was performed 1 week and 2 weeks after the initiation of treatment, while tumor volumes and weights were measured 3 weeks after the treatment. In vivo fluorescence imaging signals obtained by the uptake of ApoPep-1 to tumor was most remarkable in the group injected with cyclic form of ApoPep-1 at 1 week after combined treatment with cisplatin plus cetuximab. Correlation analysis revealed that imaging signals by cyclic ApoPep-1 at 1 week after treatment with cisplatin plus cetuximab in combination were most closely related with tumor volume changes (r2â=â0.934). These results demonstrate that in vivo apoptosis imaging using Apopep-1, especially cyclic ApoPep-1, is a sensitive and predictive tool for early decision on stomach tumor response after anti-cancer treatment.