RESUMO
Poly(3-hydroxybutyrate) (PHB) is a versatile thermoplastic with superior biodegradability and biocompatibility that is intracellularly accumulated by numerous bacterial and archaeal species. Priestia sp. strain JY310 that was able to efficiently biotransform reducing sugars in d-xylose-rich rice husk hydrolysate (reducing sugarRHH) to PHB was isolated from the soil of a rice paddy. Reducing sugarRHH including 12.5% d-glucose, 75.3% d-xylose, and 12.2% d-arabinose was simply prepared using thermochemical hydrolysis of 3% H2SO4-treated rice husk for 15 min at 121 °C. When cultured with 20 g/L reducing sugarRHH under optimized culture conditions in a batch bioreactor, Priestia sp. strain JY310 could produce PHB homopolymer up to 50.4% of cell dry weight (6.2 g/L). The melting temperature, heat of fusion, and thermal decomposition temperature of PHB were determined to be 167.9 °C, 92.1 J/g, and 268.1 °C, respectively. The number average and weight average molecular weights of PHB with a broad polydispersity index value (4.73) were estimated to be approximately 16.2 and 76.8 kg/mol, respectively. The findings of the present study suggest that Priestia sp. strain JY310 can be exploited as a good candidate for the low-cost production of low molecular weight PHB with improved biodegradability and reduced brittleness from inexpensive agricultural waste hydrolysates.
Assuntos
Bacillaceae , Oryza , Ácido 3-Hidroxibutírico , Xilose/metabolismo , Solo , Hidroxibutiratos/metabolismo , Oryza/metabolismo , Peso Molecular , Bacillaceae/metabolismo , Bactérias/metabolismo , BiotransformaçãoRESUMO
Endo-type chitinase is the principal enzyme involved in the breakdown of N-acetyl-d-glucosamine-based oligomeric and polymeric materials through hydrolysis. The gene (966-bp) encoding a novel endo-type chitinase (ChiJ), which is comprised of an N-terminal chitin-binding domain type 3 and a C-terminal catalytic glycoside hydrolase family 19 domain, was identified from a fibrolytic intestinal symbiont of the earthworm Eisenia fetida, Cellulosimicrobium funkei HY-13. The highest endochitinase activity of the recombinant enzyme (rChiJ: 30.0 kDa) toward colloidal shrimp shell chitin was found at pH 5.5 and 55 °C and was considerably stable in a wide pH range (3.5-11.0). The enzyme exhibited the highest biocatalytic activity (338.8 U/mg) toward ethylene glycol chitin, preferentially degrading chitin polymers in the following order: ethylene glycol chitin > colloidal shrimp shell chitin > colloidal crab shell chitin. The enzymatic hydrolysis of N-acetyl-ß-d-chitooligosaccharides with a degree of polymerization from two to six and colloidal shrimp shell chitin yielded primarily N,N'-diacetyl-ß-d-chitobiose together with a small amount of N-acetyl-d-glucosamine. The high chitin-degrading ability of inverting rChiJ with broad pH stability suggests that it can be exploited as a suitable biocatalyst for the preparation of N,N'-diacetyl-ß-d-chitobiose, which has been shown to alleviate metabolic dysfunction associated with type 2 diabetes.
Assuntos
Actinobacteria , Quitinases , Animais , Diabetes Mellitus Tipo 2 , OligoquetosRESUMO
Nanophotosensitizer composed of methoxy poly(ethylene glycol) (MePEG) and chlorin e6 (Ce6) (abbreviated as Pe6) was synthesized for efficient delivery of Ce6 to the colon cancer cells. Pe6 nanophotosensitizer has small diameter less than 100 nm with spherical shape and core-shell structure. They were activated in aqueous solution while Ce6 was quenched due to its poor aqueous solubility. They showed no intrinsic cytotoxicity against normal cells and colon cancer cells. Pe6 nanophotosensitizers showed enhanced cellular uptake, phototoxicity, and reactive oxygen species (ROS) generation at in vitro cell culture experiment. Furthermore, they showed improved tumor tissue penetration and accumulation in vivo animal studies. We suggested Pe6 nanophotosensitizers as an ideal candidate for PDT of colon cancer.
Assuntos
Neoplasias do Colo/terapia , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas , Animais , Linhagem Celular Tumoral , Clorofilídeos , Nanocompostos , PolietilenoglicóisRESUMO
Analysis of mixed microbial populations responsible for the production of medium-chain-length polyhydroxyalkanoates (MCL-PHAs) under periodic substrate feeding in a sequencing batch reactor (SBR) was conducted. Regardless of activated sludge samples and the different MCL alkanoic acids used as the sole external carbon substrate, denaturing gradient gel electrophoresis analysis indicated that Pseudomonas aeruginosa was the dominant bacterium enriched during the SBR process. Several P. aeruginosa strains were isolated from the enriched activated sludge samples. The isolates were subdivided into two groups, one that produced only MCL-PHAs and another that produced both MCL- and short-chain-length PHAs. The SBR periodic feeding experiments with five representative MCL-PHA-producing Pseudomonas species revealed that P. aeruginosa has an advantage over other species that enables it to become dominant in the bacterial community.
Assuntos
Bactérias/metabolismo , Poli-Hidroxialcanoatos/biossíntese , Esgotos/microbiologia , Aerobiose , Bactérias/enzimologia , Bactérias/genética , Biodiversidade , Reatores Biológicos/microbiologia , Carbono/metabolismo , Técnicas de Cultura de Células , Meios de Cultura/química , DNA Bacteriano/genética , Ácidos Graxos/metabolismo , Microbiota , Pseudomonas/classificação , Pseudomonas/enzimologia , Pseudomonas/genética , Pseudomonas/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , RNA Ribossômico 16S/genética , TemperaturaRESUMO
OBJECTIVES: To evaluate the biocatalytic characteristics of a new endo-ß-1,4-D-mannan-degrading enzyme (ManP) from Paenibacillus sp. strain HY-8, a gut bacterium of the longicorn beetle Moechotypa diphysis. RESULTS: Purified ManP (32 kDa) with an N-terminal amino acid sequence of APSFAVGADFSYVPG displayed the greatest degree of biocatalytic activity toward locust bean gum (LBG) at 55 °C and pH 7.0. The enzyme degraded LBG, guar gum, ivory nut mannan, and mannooligosaccharides (M2-M5), but did not exhibit any hydrolytic activity against structurally unrelated substrates. The biocatalytic activity of ManP against LBG and guar gum was 695 and 450 U mg-1, respectively. Especially, enzymatic hydrolysis of mannobiose yielded a mixture of mannose (16.6 %) and mannobiose (83.4 %), although the degree of mannobiose degradation by ManP with was relatively limited. CONCLUSION: The present results suggest that ManP is an endo-ß-1,4-mannanase and is distinct from various other characterized endo-ß-1,4-mannanases.
Assuntos
Paenibacillus/enzimologia , Concentração de Íons de Hidrogênio , Hidrólise , Manosidases/genética , Manosidases/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
We synthesized methoxy poly(ethylene glycol) (MPEG)-chlórin e6 (Ce6) conjugates to increase aqueous solubility of Ce6, to fabricate nanoparticles, and to improve tumor targetability of Ce6. MPEG-Ce6 conjugates (abbreviated as Pe6) associated in the aqueous solution as a nanoparticle. Pe6 nanoparticles have small diameter less than 100 nm, spherical shape, and core-shell structure in the aqueous environment. They have improved photophysical properties compared to Ce6 itself. Photosensitivity of Pe6 nanoparticles were studied using HCT116 human colon carcinoma cells. Pe6 nanoparticles practically have no dark-toxicity against HCT116 human colon carcinoma cells while they showed enhanced cellular uptake, phototoxicity, and ROS generation at in vitro cell culture experiment. Furthermore, they showed improved tumor tissue penetration and accumulation in vivo animal studies. We suggested Pe6 nanoparticles as an ideal candidate for PDT of colon cancer.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Nanopartículas/química , Fármacos Fotossensibilizantes , Polietilenoglicóis , Porfirinas , Animais , Linhagem Celular Tumoral , Clorofilídeos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Humanos , Camundongos , Camundongos Nus , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacocinética , Fármacos Fotossensibilizantes/farmacologia , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Polietilenoglicóis/farmacologia , Porfirinas/química , Porfirinas/farmacocinética , Porfirinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The gene (1608-bp) encoding a GH6 endo-ß-1,4-glucanase (CelL) from the earthworm-symbiotic bacterium Cellulosimicrobium funkei HY-13 was cloned from its whole genome sequence, expressed recombinantly, and biochemically characterized. CelL (56.0 kDa) is a modular enzyme consisting of an N-terminal catalytic GH6 domain (from Val57 to Pro396), which is 71 % identical to a GH6 protein (accession no.: WP_034662937) from Cellulomonas sp. KRMCY2, together with a C-terminal CBM 2 domain (from Cys429 to Cys532). The highest catalytic activity of CelL toward carboxymethylcellulose (CMC) was observed at 50 °C and pH 5.0, and was relatively stable at a broad pH range of 4.0-10.0. The enzyme was capable of efficiently hydrolyzing the cellulosic polymers in the order of barley ß-1,3-1,4-D-glucan > CMC > lichenan > Avicel > konjac glucomannan. However, cellobiose, cellotriose, p-nitrophenyl derivatives of mono- and disaccharides, or structurally unrelated carbohydrate polymers including ß-1,3-D-glucan, ß-1,4-D-galactomannan, and ß-1,4-D-xylan were not susceptible to CelL. The enzymatic hydrolysis of cellopentaose resulted in the production of a mixture of 68.6 % cellobiose and 31.4 % cellotriose but barley ß-1,3-1,4-D-glucan was 100 % degraded to cellotriose by CelL. The enzyme strongly bound to Avicel, ivory nut mannan, and chitin but showed relatively weak binding affinity to lichenan, lignin, or poly(3-hydroxybutyrate) granules.
Assuntos
Celulase/genética , Celulase/metabolismo , Cellulomonas/enzimologia , Oligoquetos/microbiologia , Sequência de Aminoácidos , Animais , Carboximetilcelulose Sódica/metabolismo , Celobiose/metabolismo , Celulase/química , Celulase/isolamento & purificação , Cellulomonas/genética , Quitina/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Lignina/metabolismo , Mananas/metabolismo , Dados de Sequência Molecular , Proteoglicanas , Xilanos/metabolismo , beta-Glucanas/metabolismoRESUMO
Smart delivery system of photosensitizer chlorin e6 (Ce6) has been developed for targeted photodynamic therapy (PDT). Simple self-assemblies of the mixtures comprising soybean lecithin derived phosphatidylcholine (PC), phosphatidylethanolamine-poly(L-histidine)40 (PE-p(His)40), and folic acid (FA) conjugated phosphatidylethanolamine-poly(N-isopropylacrylamide)40 (PE-p(NIPAM)40-FA) in different ratios yield smart nanospheres characterized by (i) stable and uniform particle size (â¼100 nm), (ii) positive surface charge, (iii) high hydrophobic drug (Ce6) loading efficiency up to 45%, (iv) covalently linked targeting moiety, (v) low cytotoxicity, and (vi) smartness showing p(His) block oriented pH and p(NIPAM) oriented temperature responsiveness. The Ce6-encapsulated vesicular nanospheres (Ce6@VNS) were used to confirm the efficiency of cellular uptake, intracellular distribution, and phototoxicity against KB tumor cells compared to free Ce6 at different temperature and pH conditions. The Ce6@VNS system showed significant photodynamic therapeutic efficiency on KB cells than free Ce6. A receptor-mediated inhibition study proved the site-specific delivery of Ce6 in targeted tumor cells.
Assuntos
Nanosferas/administração & dosagem , Nanosferas/química , Neoplasias/tratamento farmacológico , Polímeros/administração & dosagem , Polímeros/química , Acrilamidas/química , Linhagem Celular Tumoral , Clorofilídeos , Histidina/química , Humanos , Células KB , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/química , Porfirinas/químicaRESUMO
A series of temperature-responsive lipopolymers have been synthesized by bioconjugating poly(N-isopropylacrylamide)n (n = 25, 40, 60) onto three different phospholipids by the combination of reversible addition fragmentation chain transfer polymerization and azide-alkyne click reactions. To achieve the active targeting of cancer cells, folic acid (FA) has also been tethered to the resulting hybrid materials. The doxorubicin (Dox) encapsulated uniform nanocarriers (150 nm in diameter) fabricated by the self-assembly of the lipopolymers display temperature responsive controlled release. The FA receptor-mediated delivery of Dox was then assessed using KB cell lines, and the anti-cancer activity was assessed by the blocking of folic acid receptors. The FA-tethered lipopolymers showing temperature-responsiveness are advantageous for the cell-specific release of Dox, potentiating their anti-cancer activity.
RESUMO
Block copolymers composed of poly(3-hydroxyoctanoate) (PHO) and methoxy poly(ethylene glycol) (PEG) were synthesized to prepare paclitaxel-incorporated nanoparticle for antitumor drug delivery. In a (1)H-NMR study, chemical structures of PHO/PEG block copolymers were confirmed and their molecular weight (M.W.) was analyzed with gel permeation chromatography (GPC). Paclitaxel as a model anticancer drug was incorporated into the nanoparticles of PHO/PEG block copolymer. They have spherical shapes and their particle sizes were less than 100 nm. In a (1)H-NMR study in D2O, specific peaks of PEG solely appeared while peaks of PHO disappeared, indicating that nanoparticles have core-shell structures. The higher M.W. of PEG decreased loading efficiency and particle size. The higher drug feeding increased drug contents and average size of nanoparticles. In the drug release study, the higher M.W. of PEG block induced the acceleration of drug release rate. The increase in drug contents induced the slow release rate of drug. In an antitumor activity study in vitro, paclitaxel nanoparticles have practically similar anti-proliferation activity against HCT116 human colon carcinoma cells. In an in vivo animal study using HCT116 colon carcinoma cell-bearing mice, paclitaxel nanoparticles have enhanced antitumor activity compared to paclitaxel itself. Therefore, paclitaxel-incorporated nanoparticles of PHO/PEG block copolymer are a promising vehicle for antitumor drug delivery.
RESUMO
In this study, the effect of chlorin e6-based photodynamic therapy (Ce6-PDT) was investigated in human intrahepatic (HuCC-T1) and extrahepatic (SNU1196) cholangiocarcinoma (CCA) cells. The amount of intracellular Ce6 increased with increasing Ce6 concentration administered, or with incubation time, in both cell lines. The ability to take up Ce6 and generate reactive oxygen species after irradiation at 1.0 J/cm(2) did not significantly differ between the two CCA cell types. However, after irradiation, marked differences were observed for photodamage and apoptotic/necrotic signals. HuCC-T1 cells are more sensitive to Ce6-PDT than SNU1196 cells. Total glutathione (GSH) levels, glutathione peroxidase and glutathione reductase activities in SNU1196 cells were significantly higher than in HuCC-T1 cells. With inhibition of enzyme activity or addition of GSH, the phototoxic effect could be controlled in CCA cells. The intracellular level of GSH is the most important determining factor in the curative action of Ce6-PDT against tumor cells.
Assuntos
Colangiocarcinoma/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/farmacologia , Porfirinas/uso terapêutico , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Clorofilídeos , Colangiocarcinoma/patologia , Relação Dose-Resposta a Droga , Humanos , Fármacos Fotossensibilizantes/administração & dosagem , Porfirinas/administração & dosagem , Relação Estrutura-AtividadeRESUMO
Biocompatible lipo-histidine hybrid materials conjugated with IR820 dye show pH-sensitivity, efficient intracellular delivery of doxorubicin (Dox), and intrinsic targetability to cancer cells. These new materials form highly uniform Dox-loaded nanosized vesicles via a self-assembly process showing good stability under physiological conditions. The Dox-loaded micelles are effective for suppressing MCF-7 tumors, as demonstrated in vitro and in vivo. The combined mechanisms of the EPR effect, active internalization, endosomal-triggered release, and drug escape from endosomes, and a long blood circulation time, clearly prove that the IR820 lipopeptide DDS is a safe theranostic agent for imaging-guided cancer therapy.
Assuntos
Histidina/química , Concentração de Íons de Hidrogênio , Humanos , Células MCF-7 , Microscopia Eletrônica de TransmissãoRESUMO
Intracellular protoporphyrin IX (PpIX) generation following administration of 5-aminolevulinic acid (ALA) has been used in photodynamic therapy (PDT). Subsequent irradiation can lead to selective damage to photosensitizer-treated cells or tissues. In the present work, we describe the enhancement of ALA-induced PpIX accumulation using a liposome carrier. ALA-containing liposomes (Lipo-ALA) were prepared using dipalmitoyl-phosphatidyl choline and in vitro PDT effect was investigated against human cholangiocarcinoma HuCC-T1 cells. Lipo-ALA increased the uptake efficiency into tumor cells compared to ALA itself, which increased the phototoxic effect. A positive relationship was evident between small particle size, PpIX accumulation and cell death after Lipo-ALA based PDT.
Assuntos
Ácido Aminolevulínico/uso terapêutico , Colangiocarcinoma/tratamento farmacológico , Fotoquimioterapia , Ácido Aminolevulínico/administração & dosagem , Apoptose , Linhagem Celular Tumoral , Separação Celular , Colangiocarcinoma/patologia , Citometria de Fluxo , Humanos , LipossomosRESUMO
We fabricated cisplatin-incorporated nanoparticles using block copolymer composed of methoxy poly(ethylene glycol) (MPEG) and poly(L-glutamic acid) (PGA) (abbreviated as GE). For synthesis of block copolymer, MPEG was directly conjugated to the terminal amine of PGA. Cisplatin-incorporated nanoparticles was prepared by ion complex formation of cisplatin and PGA domain of block copolymer. Size of cisplatin-incorporated nanoparticles was less than 200 nm at particle size measurement and their shapes were spherical at TEM observation. To study drug release properties, cispaltin release from nanoparticles were performed at phosphate-buffered saline (PBS, 0.01 M, pH 7.4) and distilled water, indicating that cisplatin release rate at PBS was approximately three times higher than that of deionized water. The cell growth inhibition of cisplatin incorporated nanoparticles in vitro was tested with HuCC-T1 cells. Cisplatin-incorporated nanoparticles were effectively inhibited tumor cell growth as similar as cisplatin itself. In other words, nanoparticles showed decreased inherent cytotoxicity compared to cisplatin against normal cells. We suggest that cisplatin incorporated GE nanoparticles is a good candidate for antitumor drug targeting.
Assuntos
Antineoplásicos/química , Cisplatino/química , Nanopartículas , Polietilenoglicóis/química , Ácido Poliglutâmico/análogos & derivados , Linhagem Celular Tumoral , Humanos , Microscopia Eletrônica de Transmissão , Ácido Poliglutâmico/química , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
PURPOSE: Nanoparticles based on stimuli-sensitive drug delivery have been extensively investigated for tumor targeting. Among them, pH-responsive drug targeting using pH-sensitive polymers has attracted attention because solid tumors have an acidic environment. A dextran-b-poly(L-histidine) (DexPHS) copolymer was synthesized and pH-responsive nanoparticles were fabricated for drug targeting. METHODS AND RESULTS: A DexPHS block copolymer was synthesized by attaching the reductive end of dextran to the amine groups of poly(L-histidine). pH-responsive nanoparticles incorporating doxorubicin were fabricated and studied in HuCC-T1 cholangiocarcinoma cells. Synthesis of DexPHS was confirmed by 1H nuclear magnetic resonance spectroscopy, with specific peaks of dextran and PHS observed at 2-5 ppm and 7.4-9.0 ppm, respectively. DexPHS nanoparticles showed changes in particle size with pH sensitivity, ie, the size of the nanoparticles increased at an acidic pH and decreased at a basic pH. DexPHS block copolymer nanoparticles incorporating doxorubicin were prepared using the nanoprecipitation dialysis method. The doxorubicin release rate was increased at acidic pH compared with basic pH, indicating that DexPHS nanoparticles have pH-sensitive properties and that drug release can be controlled by variations in pH. The antitumor activity of DexPHS nanoparticles incorporating doxorubicin were studied using HuCC-T1 cholangiocarcinoma cells. Viability was decreased in cells treated with nanoparticles at acidic pH, whereas cell viability in response to treatment with doxorubicin did not vary according to changes of pH. CONCLUSION: Our results indicated that DexPHS polymeric micelles are promising candidates for antitumor drug targeting.
Assuntos
Antineoplásicos/farmacocinética , Dextranos/química , Portadores de Fármacos/farmacocinética , Histidina/química , Nanopartículas/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Humanos , Concentração de Íons de Hidrogênio , Nanomedicina , Nanopartículas/toxicidade , Tamanho da Partícula , Polímeros/químicaRESUMO
Chitosan was hydrophobically modified with ursodeoxycholic acid (UDCA) to fabricate nano-photosensitizer for photodynamic therapy (PDT) of HuCC-T1 cholangiocarcinoma cells. Synthesis of UDCA-conjugated chitosan (ChitoUDCA) was confirmed using (1)H NMR spectra. Chlorin E6 (Ce6) was used as a photosensitizer and incorporated into ChitoUDCA nanoparticles through formation of ion complexes. Morphology of Ce6-incorporated ChitoUDCA nanoparticles was observed using TEM and their shapes were spherical with sizes around 200-400 nm. The PDT potential of Ce6-incorporated ChitoUDCA nanoparticles were studied with HuCC-T1 human cholangiocarcinoma cells. The results showed that ChitoUDCA nanoparticles enhances of Ce6 uptake into tumor cells, phototoxicity, and ROS generation compared to Ce6 itself. Furthermore, Ce6-incorporated ChitoUDCA nanoparticles showed quenching in aqueous solution and sensing at tumor cells. We suggest that Ce6-incorporated ChitoUDCA nanoparticles are promising candidates for PDT of cholangiocarcinoma cells.
Assuntos
Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/efeitos dos fármacos , Quitosana/química , Colangiocarcinoma/patologia , Portadores de Fármacos , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Ácido Ursodesoxicólico/metabolismo , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Transporte Biológico , Linhagem Celular Tumoral , Química Farmacêutica , Clorofilídeos , Colangiocarcinoma/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Fármacos Fotossensibilizantes/química , Porfirinas/química , Espécies Reativas de Oxigênio/metabolismo , Tecnologia Farmacêutica/métodos , Ácido Ursodesoxicólico/químicaRESUMO
The aim of this study was to investigate the effect of the combination of vorinostat and epigallocatechin-3-gallate against HuCC-T1 human cholangiocarcinoma cells. A novel chemotherapy strategy is required as cholangiocarcinomas rarely respond to conventional chemotherapeutic agents. Both vorinostat and EGCG induce apoptosis and suppress invasion, migration, and angiogenesis of tumor cells. The combination of vorinostat and EGCG showed synergistic growth inhibitory effects and induced apoptosis in tumor cells. The Bax/Bcl-2 expression ratio and caspase-3 and -7 activity increased, but poly (ADP-ribose) polymerase expression decreased when compared to treatment with each agent alone. Furthermore, invasion, matrix metalloproteinase (MMP) expression, and migration of tumor cells decreased following treatment with the vorinostat and EGCG combination compared to those of vorinostat or EGCG alone. Tube length and junction number of human umbilical vein endothelial cells (HUVECs) decreased as well as vascular endothelial growth factor expression following vorinostat and EGCG combined treatment. These results indicate that the combination of vorinostat and EGCG had a synergistic effect on inhibiting tumor cell angiogenesis potential. We suggest that the combination of vorinostat and EGCG is a novel option for cholangiocarcinoma chemotherapy.
RESUMO
5-Aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has the potential to kill cancer cells via apoptotic or necrotic signals that are dependent on the generation of intracellular reactive oxygen species (ROS). Celecoxib is an anti-inflammatory drug that induces intracellular ROS generation. We investigated whether the combined application of celecoxib and ALA-PDT improved the efficacy of PDT in human cholangiocarcinoma cells and in tumor bearing mice. In vitro, combined treatment of celecoxib and ALA-PDT increased phototoxicity and intracellular ROS levels after irradiation with 0.75 J/cm(2) when compared to ALA-PDT alone. Even though ROS levels increased with 0.25 J/cm(2) of irradiation, it did not influence phototoxicity. When heme oxygenase-1, a defensive protein induced by oxidative stress, was inhibited in the combined treatment group, phototoxicity was increased at both 0.25 J/cm(2) and 0.75 J/cm(2) of irradiation. We identified the combined effect of ALA-PDT and celecoxib through the increase of oxidative stress such as ROS. In vivo, about 40% tumor growth inhibition was observed with combined application of ALA-PDT and celecoxib when compared to ALA-PDT alone. The combined application of ALA-PDT and celecoxib could be an effective therapy for human cholangiocarcinoma. Moreover, use of a heme oxygenase-1 inhibitor with PDT could play an important role for management of various tumors involving oxidative stress.
Assuntos
Ácido Aminolevulínico/farmacologia , Colangiocarcinoma , Estresse Oxidativo/efeitos dos fármacos , Fotoquimioterapia/métodos , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Ácido Aminolevulínico/química , Análise de Variância , Animais , Neoplasias dos Ductos Biliares , Ductos Biliares Intra-Hepáticos , Celecoxib , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Heme Oxigenase-1/análise , Heme Oxigenase-1/metabolismo , Humanos , Masculino , Camundongos Endogâmicos BALB C , Pirazóis/química , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Sulfonamidas/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Cholangiocarcinoma is a malignant tumor arising from the epithelium of the bile ducts. In this study, we prepared sorafenib-loaded biliary stents for potential application as drug-delivery systems for localized treatment of extrahepatic cholangiocarcinoma. METHODS: A sorafenib-coated metal stent was prepared using an electrospray system with the aid of poly(ε-caprolactone) (PCL), and then its anticancer activity was investigated using human cholangiocellular carcinoma (HuCC)-T1 cells in vitro and a mouse tumor xenograft model in vivo. Anticancer activity of sorafenib against HuCC-T1 cells was evaluated by the proliferation test, matrix metalloproteinase (MMP) activity, cancer cell invasion, and angiogenesis assay in vitro and in vivo. RESULTS: The drug-release study showed that the increased drug content on the PCL film induced a faster drug-release rate. The growth of cancer cells on the sorafenib-loaded PCL film surfaces decreased in a dose-dependent manner. MMP-2 expression of HuCC-T1 cells gradually decreased according to sorafenib concentration. Furthermore, cancer cell invasion and tube formation of human umbilical vein endothelial cells significantly decreased at sorafenib concentrations higher than 10 mM. In the mouse tumor xenograft model with HuCC-T1 cells, sorafenib-eluting PCL films significantly inhibited the growth of tumor mass and induced apoptosis of tumor cells. Various molecular signals, such as B-cell lymphoma (Bcl)-2, Bcl-2-associated death promoter, Bcl-x, caspase-3, cleaved caspase-3, Fas, signal transducer and activator of transcription 5, extracellular signal-regulated kinases, MMP-9 and pan-janus kinase/stress-activated protein kinase 1, indicated that apoptosis, inhibition of growth and invasion was cleared on sorafenib-eluting PCL films. CONCLUSION: These sorafenib-loaded PCL films are effective in inhibiting angiogenesis, proliferation and invasion of cancer cells. We suggest that sorafenib-loaded PCL film is a promising candidate for the local treatment of cholangiocarcinoma.
Assuntos
Antineoplásicos/farmacologia , Neoplasias dos Ductos Biliares , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma , Stents Farmacológicos , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Nus , Niacinamida/química , Niacinamida/farmacocinética , Niacinamida/farmacologia , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacocinética , Poliésteres , Sorafenibe , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of dual stimuli responsive synthetic polymer bioconjugate chimeric materials, poly(N-isopropylacrylamide)55-block-poly(L-histidine)n [p(NIPAM)55-b-p(His)n] (n=50, 75, 100, 125), have been synthesized by employing reversible addition-fragmentation chain transfer polymerization of NIPAM, followed by ring-opening polymerization of α-amino acid N-carboxyanhydrides. The dual stimuli responsive properties of the resulting biocompatiable and membrenolytic p(NIPAM)55-b-p(His)n polymers are investigated for their use as a stimuli responsive drug carrier for tumor targeting. Highly uniform self-assembled micelles (â¼55 nm) fabricated by p(NIPAM)55-b-p(His)n polymers display sharp thermal and pH responses in aqueous media. An anticancer drug, doxorubicin (Dox), is effectively encapsulated in the micelles and the controlled Dox release is investigated in different temperature and pH conditions. Antitumor effect of the released Dox is also assessed using the HepG2 human hepatocellular carcinoma cell lines. Dox molecules released from the [p(NIPAM)55-b-p(His)n] micelles remain biologically active and have stimuli responsive capability to kill cancer cells. The self-assembling ability of these hybrid materials into uniform micelles and their efficiency to encapsulate Dox makes them a promising drug carrier to cancer cells. The new chimeric materials thus display tunable properties that can make them useful for a molecular switching device and controlled drug delivery applications needing responses to temperature and pH for the improvement of cancer chemotherapy.