RESUMO
BACKGROUND: Numerous researches have focused on discovering available inhibitors of melanogenesis from natural medicinal plants with stable efficacy and safety to resolve cutaneous hyperpigmentary problems. Melochia corchorifolia Linn. (MC) has been used as folk medicine to treat various diseases. However, the effect of MC on melanogenesis remains unknown. AIM: In this study, we investigated the effect of MC extract on melanogenesis and its underlying mechanisms in B16F10 mouse melanoma cells. METHODS: B16F10 cells were treated with MC extract, and then, cell viability, melanin content, and tyrosinase activity were analyzed. The mRNA and protein expression of tyrosinase and microphthalmia-associated transcription factor (MITF) were evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting, respectively. Phosphorylated or total protein levels in MC extract-induced signaling pathways were analyzed by Western blotting. RESULTS: Treatment of B16F10 cells with MC extract inhibited melanin synthesis and intracellular tyrosinase activity in a dose-dependent manner with no cytotoxicity. Protein and mRNA expressions of tyrosinase and MITF were also significantly decreased by MC extract treatment. In addition, phosphorylated level of extracellular signal-regulated kinase (ERK) was obviously increased by MC extract, but AKT pathway was not activated. Inhibited ERK phosphorylation by pretreatment with a selective ERK inhibitor PD98059 significantly reversed the decreased melanin content induced by treatment with MC extract in B16F10 cells. CONCLUSION: MC extract inhibits melanogenesis in B16F10 mouse melanoma cells through suppression of MITF-tyrosinase signaling pathway by ERK activation.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular , Malvaceae/química , Melanoma Experimental , Extratos Vegetais/uso terapêutico , Transdução de Sinais , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Melaninas , Melanoma Experimental/tratamento farmacológico , Camundongos , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismoRESUMO
Keloids were characterized by excessive growth of fibrous tissues, and shared several pathological characteristics with cancer. They did put physical and emotional stress on patients in that keloids could badly change appearance of patients. N-(4-hydroxyphenyl) retinamide (4HPR) showed cytotoxic activity on a wide variety of invasive-growth cells. Our work was aim to prepare N-(4-hydroxyphenyl) retinamide-loaded lipid microbubbles (4HPR-LM) combined with ultrasound for anti-keloid therapy. 4HPR-loaded liposomes (4HPR-L) were first prepared by film evaporation method, and then 4HPR-LM were manufactured by mixing 4HPR-L and perfluoropentane (PFP) with ultrasonic cavitation method. The mean particle size and entrapment efficiency 4HPR-LM were 113 nm and 95%, respectively. The anti-keloids activity of 4HPR-LM was assessed with BALB/c nude mice bearing subcutaneous xenograft keloids model. 4HPR-LM, combined with ultrasound, could significantly induce apoptosis of keloid fibroblasts in vitro and inhibited growth of keloids in vivo. Thus, 4HPR-LM could be considered as a promising agent for anti-keloids therapy.
Assuntos
Fenretinida/farmacologia , Queloide/terapia , Lipídeos/química , Lipossomos , Nanopartículas , Ondas Ultrassônicas , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fenretinida/química , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Vasoactive intestinal peptide (VIP), one of the major skin neuropeptides, has been suggested to have active roles in the pathogenesis of inflammatory skin disorders such as atopic dermatitis and psoriasis, which can commonly cause post-inflammatory hyperpigmentation. However, the effect of VIP on melanogenesis remains unknown. In this study, we showed that the melanin contents, tyrosinase activity, and gene expression of tyrosinase and microphthalmia-associated transcription factor (MITF) were significantly increased by treatment with VIP in B16F10 mouse melanoma cells and the stimulatory melanogenic effect was further examined in human epidermal melanocytes (HEMns). In addition, phosphorylated levels of CRE-binding protein (CREB) and protein kinase A (PKA) were markedly increased after VIP treatment, but not p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), or Akt, indicating the possible PKA-CREB signaling pathway involved in VIP-induced melanogenesis. This result was further verified by the fact that VIP induced increased melanin synthesis, and protein levels of phosphorylated CREB, MITF, tyrosinase were significantly attenuated by H89 (a specific PKA inhibitor). These data suggest that VIP-induced upregulation of tyrosinase through the CREB-MITF signaling pathway plays an important role in finding new treatment strategy for skin inflammatory diseases related pigmentation disorders.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Melaninas/biossíntese , Melanoma Experimental/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Transdução de Sinais , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Melanoma Experimental/patologia , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , RNA Mensageiro/genéticaRESUMO
Neuronal nitric oxide synthase (nNOS) is important in cardiac protection in diseased heart. Recently, we have reported that nNOS is associated with myofilament Ca(2+) desensitization in cardiac myocytes from hypertensive rats. So far, the effect of myofilament Ca(2+) desensitization or nNOS on L-type Ca(2+) channel activity (I(Ca)) in cardiac myocyte is unclear. Here, we examined nNOS regulation of I(Ca) in left ventricular (LV) myocytes from sham and angiotensin II (Ang II)-induced hypertensive rats. Our results showed that basal I(Ca) was not different between sham and hypertension (from -60 to +40 mV, 0.1 Hz). S-methyl-L-thiocitrulline (SMTC), a selective nNOS inhibitor, increased peak I(Ca) similarly in both groups. However, chelation of intracellular Ca(2+) [Ca(2+)]i with BAPTA increased I(Ca) and abolished SMTC-augmentation of I(Ca) only in hypertension. Myofilament Ca(2+) desensitization with butanedione monoxime (BDM), a myosin ATPase inhibitor, decreased I(Ca) in both groups but to a greater extent in hypertension. Intracellular BAPTA or nNOS inhibition reinstated I(Ca) in the presence of BDM to the basal level, suggesting Ca(2+)-dependent inactivation of I(Ca) by nNOS and greater vulnerability in hypertension. Increasing stimulation frequencies (2, 4 and 8 Hz) attenuated myofilament Ca(2+) sensitivity in sham and reduced peak ICa in both groups. Nevertheless, SMTC or BAPTA exerted no effect on I(Ca) at high frequencies in either group. These results suggest that nNOS attenuates I(Ca) via Ca(2+)-dependent mechanism and the vulnerability is greater in hypertension subject to myofilament Ca(2+) desensitization. nNOS or [Ca(2+)]i does not affect I(Ca) at high stimulation frequencies. The results were recapitulated with computer simulation.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Sinalização do Cálcio , Hipertensão/metabolismo , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Canais de Cálcio , Ventrículos do Coração/metabolismo , Hipertensão/induzido quimicamente , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Biglycan (BGN) is a proteoglycan composed of a 42-kDa core protein and two glycosaminoglycan (GAG) chains, and known to be involved in structural, space-filling functions and many physiological regulations in the skin. OBJECTIVE: To investigate ultraviolet (UV) irradiation-induced changes of BGN protein and its GAG chain synthesis in cultured human dermal fibroblasts. METHODS: UV irradiation-induced or xylosyltransferase (XYLT) 1 siRNA-mediated smaller-sized protein bands detected by Western blot using BGN antibodies were identified as monoglycosylated forms of BGN, using BGN siRNA-mediated knockdown and chondroitinase ABC (ChABC). Differential activity of XYLT1 and 2 on BGN core protein was investigated by size shift of S42A- and S47A-BGN mutants to core protein size caused by XYLT1 siRNA transfection or UV irradiation. RESULTS: After UV irradiation, intact form of BGN protein (I-BGN) and core protein form were reduced in cultured fibroblasts, but other smaller-sized bands were observed to be increased. These smaller-sized ones were reduced by transfection of BGN siRNA, and shifted to the core protein size by treatment with ChABC, suggesting that they are defectively-glycosylated forms of BGN (D-BGN) protein. UV irradiation also decreased mRNA expression levels of XYLT1 and 2, which are responsible for initiation of GAG chain synthesis. UV-mediated reduction of XYLT1 expression was much stronger than that of XYLT2. Furthermore, siRNA-mediated down-regulation of XYLT1 resulted in the increase of D-BGN and the decrease of I-BGN, while down-regulation of XYLT2 resulted in no change of D-BGN and I-BGN, suggesting that the XYLT1 may react with both GAG-attaching serine sites of BGN; however, XYLT2 may prefer to react one of them. Another dermatan sulfate (DS) proteoglycan, decorin, showed no or a little change of its molecular weight by UV irradiation or XYLT1 siRNA transfection, suggesting that DS synthesis may not be a critical factor in formation of D-BGN. Co-transfection with XYLT1, 2 siRNAs and wild-type or mutant forms of BGN overexpression vectors revealed that S42A-BGN showed size reduction to core protein size by XYLT1 downregulation, but S47A-BGN did not, suggesting that XYLT2 can react only with S42 on BGN core protein. With UV irradiation, both S42A-BGN and S47A-BGN showed size reduction, which is probably because UV-caused downregulation of both XYLTs and overexpression condition resulted in incomplete glycosylation and secretion. CONCLUSIONS: UV irradiation-induced increase of BGN monoglycosylated forms in cultured human dermal fibroblasts is resulted from dominance of XYLT2 activity, which acts only at S42 on BGN core protein, caused by UV-mediated stronger reduction of XYLT1.
Assuntos
Biglicano/biossíntese , Biglicano/genética , Glicosaminoglicanos/biossíntese , Pentosiltransferases/metabolismo , Raios Ultravioleta , Células Cultivadas , Decorina/metabolismo , Regulação para Baixo/efeitos da radiação , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Inativação Gênica , Glicosaminoglicanos/efeitos da radiação , Glicosilação/efeitos da radiação , Humanos , Peso Molecular , Pentosiltransferases/genética , Pentosiltransferases/efeitos da radiação , Biossíntese de Proteínas/efeitos da radiação , RNA Mensageiro/metabolismo , Fenômenos Fisiológicos da Pele/efeitos da radiação , Xilose/metabolismo , UDP Xilose-Proteína XilosiltransferaseRESUMO
Angiotensin II (Ang II), a potent precursor of hypertrophy and heart failure, upregulates neuronal nitric oxide synthase (nNOS or NOS1) in the myocardium. Here, we investigate the involvement of type 1 and 2 angiotensin receptors (AT1R and AT2R) and molecular mechanisms mediating Ang II-upregulation of nNOS. Our results showed that pre-treatment of left ventricular (LV) myocytes with antagonists of AT1R or AT2R (losartan, PD123319) and ROS scavengers (apocynin, tiron or PEG-catalase) blocked Ang II-upregulation of nNOS. Surface biotinylation or immunocytochemistry experiments demonstrated that AT1R expression in plasma membrane was progressively decreased (internalization), whereas AT2R was increased (membrane trafficking) by Ang II. Inhibition of AT1R or ROS scavengers prevented Ang II-induced translocation of AT2R to plasma membrane, suggesting an alignment of AT1R-ROS-AT2R. Furthermore, Ang II increased eNOS-Ser(1177) but decreased eNOS-Thr(495), indicating concomitant activation of eNOS. Intriguingly, ROS scavengers but not AT2R antagonist prevented Ang II-activation of eNOS. NOS inhibitor (L-NG-Nitroarginine Methyl Ester, L-NAME) or eNOS gene deletion (eNOS(-/-)) abolished Ang II-induced membrane trafficking of AT2R, nNOS protein expression and activity. Mechanistically, S-nitrosation of AT2R was increased by sodium nitroprusside (SNP), a NO donor. Site-specific mutagenesis analysis reveals that C-terminal cysteine 349 in AT2R is essential in AT2R translocation to plasma membrane. Taken together, we demonstrate, for the first time, that Ang II upregulates nNOS protein expression and activity via AT1R/ROS/eNOS-dependent S-nitrosation and membrane translocation of AT2R. Our results suggest a novel crosstalk between AT1R and AT2R in regulating nNOS via eNOS in the myocardium under pathogenic stimuli.
Assuntos
Angiotensina II/metabolismo , Miócitos Cardíacos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Animais , Células HEK293 , Humanos , Immunoblotting , Imuno-Histoquímica , Masculino , Microscopia Confocal , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Receptor Cross-Talk , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
T cell immunoglobulin domain and mucin domain-containing molecule 3 (Tim-3) is a newly discovered immunomodulatory, which plays an important role in immunity regulation. Recent evidence suggests that Tim-3 is differentially regulated in a variety of tumors and has a potential as a therapeutic target. The aim of this study was to investigate the effect of Tim-3 on the development of prostate cancer (PCa). Tim-3 expressing on peripheral CD4+ T and CD8+ T cells was analyzed by flow cytometry. The relationships between Tim-3 expression and clinicopathological features were analyzed. Immunohistochemical expression of Tim-3 was examined in our large numbers of paraffin-fixed prostate tissues. Flow cytometry revealed that expression of Tim-3 was significantly increased on both CD4+ and CD8+ T cells in PCa patients than that in benign prostate hyperplasia (BPH) patients. Also, the level of Tim-3 on CD4+ T cells was positively correlated with CD8+ T cells in patients. Further analyses revealed that the levels of Tim-3 on CD4+ T cells and CD8+ T cells exhibited different expression patterns in terms of localization depending on pathological category of PCa and metastasis. Immunohistochemical analysis revealed that positive staining of Tim-3 in PCa but little or no staining of Tim-3 was observed in BPH epithelium. Tim-3 may affect the development and progression of PCa, which may provide knowledge for using Tim-3 as a novel therapy for effective PCa management.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Proteínas de Membrana/metabolismo , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Progressão da Doença , Citometria de Fluxo , Seguimentos , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Hiperplasia Prostática/imunologia , Hiperplasia Prostática/patologia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologiaRESUMO
AIM: To evaluation the doxorubicin (DOX)-loaded pH-sensitive polymeric micelle release from tumor blood vessels into tumor interstitium using an animal vessel visibility model, the so-called dorsal skin-fold window chamber model. METHODS: DOX-loaded pH-sensitive polyHis-b-PEG micelles and DOX-loaded pH-insensitive PLLA-b-PEG micelles were prepared. The uptake of the micelles by MDA-MB-231 breast cancer cells in vitro and in vivo was examined using flow cytometry. The pharmacokinetic parameters of the micelles were determined in SD rats after intravenous injection of a DOX dose (6 mg/kg). The release of the micelles from tumor vasculature and the antitumor efficacy were evaluated in MDA-MB-231 breast cancer xenografted in nude mice using a dorsal skin-fold window chamber. RESULTS: The effective elimination half-life t1/2 of the pH-sensitive, pH-insensitive polymeric micelles and DOX-PBS in rats were 11.3 h, 9.4 h, and 2.1 h, respectively. Intravital microscopy in MDA-MB-231 breast cancer xenografted in nude mice showed that the pH-sensitive polymeric micelles rapidly extravasated from the tumor blood vessels, and DOX carried by the pH-sensitive micelles was preferentially released at the tumor site as compared to the pH-insensitive polymeric micelles. Furthermore, the pH-sensitive polymeric micelles exhibited significant greater efficacy in inhibition of tumor growth in the nude mice. CONCLUSION: When DOX is loaded into pH-sensitive polymeric micelles, the acidity in tumor interstitium causes the destabilization of the micelles and triggers drug release, resulting in high local concentrations within the tumor, thus more effectively inhibiting the tumor growth in vivo.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/tratamento farmacológico , Mama/efeitos dos fármacos , Preparações de Ação Retardada/química , Doxorrubicina/administração & dosagem , Micelas , Animais , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/uso terapêutico , Mama/irrigação sanguínea , Mama/patologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Doxorrubicina/farmacocinética , Doxorrubicina/uso terapêutico , Feminino , Humanos , Concentração de Íons de Hidrogênio , Camundongos Endogâmicos BALB C , Camundongos Nus , Polímeros/química , Próteses e Implantes , Ratos Sprague-DawleyRESUMO
This study primarily focused on the systematic assessment of both in vitro and in vivo anti-tumor effects of docetaxel-loaded polyethylene glycol (PEG)2000-polycaprolactone (PCL)2600 micelles on hormone-refractory prostate cancer (HRPC). By using solvent evaporation method, PEG-PCL was chosen to prepare doxetaxel (DTX)-loaded mPEG-PCL micelles (DTX-PMs), with the purpose of eliminating side effects of the commercial formulation (Tween 80) and prolonging the blood circulation time. The prepared DTX-PMs had an average particle size of 25.19±2.36 nm, a zeta potential of 0.64±0.15 mV, a polydispersity index of 0.56±0.03, a drug loading of (8.72±1.05)%, and an encapsulation efficiency of (98.1±8.4)%. In vitro cytotoxicity studies indicated that DTX-PMs could effectively kill LNCap-C4-2B cells and show a dose- and time-dependent efficacy. The hemolysis test showed that DTX-PMs had less hemocytolysis than the commercial product of Duopafei®. A sustained in vitro release behavior and prolonged circulation time in blood vessels were observed in the DTX-PMs. Furthermore, when compared with Duopafei®, the DTX-PMs dramatically reduced the prostate specific antigen (PSA) level and tumor growth of prostate tumor-bearing nude mice in vivo. In conclusion, the DTX-PMs can lower systemic side effects, improve anti-tumor activity with prolonged blood circulation time, and will bring an alternative to patients with HRPC.
Assuntos
Antineoplásicos/farmacologia , Micelas , Neoplasias da Próstata/tratamento farmacológico , Taxoides/farmacologia , Animais , Antineoplásicos/farmacocinética , Área Sob a Curva , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Docetaxel , Relação Dose-Resposta a Droga , Cobaias , Hemólise/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Nus , Tamanho da Partícula , Poliésteres/química , Polietilenoglicóis/química , Neoplasias da Próstata/patologia , Ratos , Ratos Sprague-Dawley , Taxoides/química , Taxoides/farmacocinética , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Optimal treatment for prostate cancer remains a challenge worldwide. Recently, T cell immunoglobulin mucin-3 (TIM-3) has been implicated in tumor biology but its contribution prostate cancer remains unclear. The aim of this study was to investigate the role of TIM-3 as a prognostic marker in patients with prostate cancer. METHODS: TIM-3 protein expression was determined by immunohistochemistry and Western blotting in 137 prostate cancer tumor samples and paired adjacent benign tissue. We also performed cell proliferation assays using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl- 2H tetrazolium bromide (MTT) and cell invasion assays. The effects of small interfering RNA (siRNA)-mediated knockdown of TIM-3 (TIM-3 siRNA) in two human prostate cancer cell lines were also evaluated. RESULTS: TIM-3 expression was higher in prostate cancer tissue than in the adjacent benign tissue (P<0.001). High TIM-3 expression was an independent predictor of both recurrence-free survival and progression-free survival. TIM-3 protein was expressed in both prostate cancer cell lines and knockdown suppressed their proliferation and invasion capacity. CONCLUSIONS: TIM-3 expression is associated with a poor prognosis in prostate cancer. Taken together, our results indicate that TIM-3 is a potential prognostic marker in prostate cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/mortalidade , Neoplasias Pulmonares/mortalidade , Proteínas de Membrana/metabolismo , Neoplasias da Próstata/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Biomarcadores Tumorais/genética , Western Blotting , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Proliferação de Células , Seguimentos , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Metástase Linfática , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Gradação de Tumores , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Prognóstico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
Neuronal nitric oxide synthase (NOS1 or nNOS) exerts negative inotropic and positive lusitropic effects through Ca(2+) handling processes in cardiac myocytes from healthy hearts. However, underlying mechanisms of NOS1 in diseased hearts remain unclear. The present study aims to investigate this question in angiotensin II (Ang II)-induced hypertensive rat hearts (HP). Our results showed that the systolic function of left ventricle (LV) was reduced and diastolic function was unaltered (echocardiographic assessment) in HP compared to those in shams. In isolated LV myocytes, contraction was unchanged but peak [Ca(2+)]i transient was increased in HP. Concomitantly, relaxation and time constant of [Ca(2+)]i decay (tau) were faster and the phosphorylated fraction of phospholamban (PLN-Ser(16)/PLN) was greater. NOS1 protein expression and activity were increased in LV myocyte homogenates from HP. Surprisingly, inhibition of NOS1 did not affect contraction but reduced peak [Ca(2+)]i transient; prevented faster relaxation without affecting the tau of [Ca(2+)]i transient or PLN-Ser(16)/PLN in HP, suggesting myofilament Ca(2+) desensitization by NOS1. Indeed, relaxation phase of the sarcomere length-[Ca(2+)]i relationship of LV myocytes shifted to the right and increased [Ca(2+)]i for 50% of sarcomere shortening (EC50) in HP. Phosphorylations of cardiac myosin binding protein-C (cMyBP-C(282) and cMyBP-C(273)) were increased and cardiac troponin I (cTnI(23/24)) was reduced in HP. Importantly, NOS1 or PKG inhibition reduced cMyBP-C(273) and cTnI(23/24) and reversed myofilament Ca(2+) sensitivity. These results reveal that NOS1 is up-regulated in LV myocytes from HP and exerts positive lusitropic effect by modulating myofilament Ca(2+) sensitivity through phosphorylation of key regulators in sarcomere.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Regulação Enzimológica da Expressão Gênica , Hipertensão/enzimologia , Miocárdio/enzimologia , Miofibrilas/enzimologia , Óxido Nítrico Sintase Tipo I/biossíntese , Animais , Células Cultivadas , Valvas Cardíacas/enzimologia , Valvas Cardíacas/patologia , Hipertensão/patologia , Camundongos , Contração Miocárdica , Miocárdio/patologia , Miofibrilas/patologia , Óxido Nítrico Sintase Tipo I/genética , Ratos , Ratos Sprague-DawleyRESUMO
Gene silencing induced by RNA interference using small interfering RNA (siRNA) provides a promising therapeutic approach for cancers. However, the lack of siRNA delivery vector has limited the development of siRNA therapy. The purpose of this study was to use the novel copolymer (mPEG5k-PCL1.2k)1.4-g-PEl10k to prepare siRNA-loaded nanoparticles for siRNA delivery. The results suggested that (mPEG5k-PCL1.2k)1.4-g-PEl10k could load siRNA to form nanoparticles with particle size less than 200 nm in a narrow distribution. Moreover, a certain density of positive charge existed onto the surfaces of nanoparticles. MTT assay results demonstrated that (mPEG5k-PCL1.2k)1.4-g-PEl10k/siRNA nanoparticles showed very low cytotoxicity. The gene silencing efficiency of (mPEG5k-PCL1.2k)1.4-g-PEl10k/siRNA nanoparticles was investigated through luciferase reporter gene assays. The expression of exogenous luciferase gene was significantly downregulated at a range of N/P ratio from 50 to 125, and was maximally inhibited at the N/P ratio of 125 with 54% and 59% reduction in MCF-7 and HepG2 cells, respectively. In the 4T1-luc cell line expressing luciferase stably, the silencing of endogenous luciferase gene also has a similar overall profile with maximal 54% reduction of luciferase expression. These results suggested that (mPEG5k-PCL1.2k)1.4-g-PEI10k/SiRNA nanoparticles could serve as a kind of highly efficient siRNA delivery system for down-regulating the expression of exogenous and endogenous target genes.
Assuntos
Sistemas de Liberação de Medicamentos , Técnicas de Transferência de Genes , Nanopartículas , Polímeros/química , RNA Interferente Pequeno/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Corantes , Regulação para Baixo/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Marcação de Genes , Humanos , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Poliésteres , Polietilenoglicóis , Sais de Tetrazólio , TiazóisRESUMO
Chemotherapy drug Doxorubicin and MRI imaging agent Iron Oxide were encapsulated in dual functional nanoparticles which is compose of PEG-Foliate acid polymers. The particle size was about 45 nm with a mono-dispersion measured by zeta-sizer instrument. The contrast of images in liver was enhanced after injection Nanoparticles via rabbit ear vein. PEG-FA coated Iron Oxide nanoparticles can selectively target to tumor site and effectively inhibit tumor growth which is subcutaneously bearing A2780 Overran cancer cells. It is feasible for cancer treatment with MRI imaging and chemotherapy simultaneously in vivo by injection dual functional nanoparticles.
Assuntos
Nanopartículas , Neoplasias Experimentais/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , CoelhosRESUMO
To improve bioavailability of poorly water-soluble YH439, a thermal reversible microemulsion system was prepared using modified fatty acids such as capric acid and palmitic acid with PEG 400. A combination of Capric-PEG 400 and Palmitic-PEG 400 with a ratio of 1 : 3 used as a lipid matrix and Cremophor RH40 and Neobee M-5 were selected as an oil and a surfactant, respectively. The microemulsion with melting point of 36.5 degrees C was produced by mixing the lipid matrices, Cremophor RH40 and Neobee M-5 with a volume ratio of 5 : 4 : 1. After the microemulsion was dispersed in the aqueous medium, the average particle size of 28 nm was obtained. At the release measurements of YH439 after 45 min suspension in pH 1.2 aqueous medium, about 80%, 65%, 10% and less than 5% of drug were released from the thermal reversible microemulson, Gelucire formulation, 5% Ca-carboxymethylcellulose (CMC) suspension and YH439 powder, respectively. The apparent permeability of YH439 in microemulsion either from apical to basolateral or basolateral to apical after measuring YH439 across a Caco-2 cell monolayer in a Transwell larger than Gelucire formulation or 5% Na-CMC suspension. The area under the drug concentration-time curves (AUC) and maximal blood concentration (C(max)) after oral administration of YH439 loaded on thermal reversible microemulsion were significantly increased than drug loaded on either Gelucire formulation or 5% Na-CMC suspension. Thus, the present work demonstrates that the thermal reversible microemulsion system of YH439 greatly enhances the bioavailability of YH439 after oral administration due to the improvement of solubility and dispersion of the drug in the artificial gastrointestinal tract without pepsin.