RESUMEN
Skin grafting is often the only treatment for skin trauma when large areas of tissue are affected. This surgical intervention damages the deeper dermal layers of the skin with implications for wound healing and a risk of scar development. Photobiomodulation (PBM) therapy modulates biological processes in different tissues, with a positive effect on many cell types and pathways essential for wound healing. This study investigated the effect of fluorescent light energy (FLE) therapy, a novel type of PBM, on healing after skin grafting in a dermal fibrotic mouse model. Split-thickness human skin grafts were transplanted onto full-thickness excisional wounds on nude mice. Treated wounds were monitored, and excised xenografts were examined to assess healing and pathophysiological processes essential for developing chronic wounds or scarring. Results demonstrated that FLE treatment initially accelerated re-epithelialization and rete ridge formation, while later reduced neovascularization, collagen deposition, myofibroblast and mast cell accumulation, and connective tissue growth factor expression. While there was no visible difference in gross morphology, we found that FLE treatment promoted a balanced collagen remodeling. Collectively, these findings suggest that FLE has a conceivable effect at balancing healing after skin grafting, which reduces the risk of infections, chronic wound development, and fibrotic scarring.
RESUMEN
Macrophages play a key role in the wound healing process and can be divided into classically activated macrophages (M1) and alternatively activated macrophages (M2). Fibroblasts maintain the physical integrity of connective tissue, participate in wound closure as well as produce and remodel extracellular matrix. Macrophages have a close relationship with fibroblasts by increasing the production of matrix metalloproteinase-1 (MMP-1) for faster wound closure and remodeling and myofibroblast differentiation from fibroblasts. In this study, resting state (M0), M1 and M2 macrophages differentiated from the human monocytic THP-1 cell line were used to co-culture with human dermal fibroblasts (HDF) for 48, 96 and 144 hours to investigate the effect of macrophages subsets on the fibrogenic activity of fibroblasts. The differentiation and polarization from THP-1 cells to M0, M1 and M2 macrophages were characterized by flow cytometry and cell cycle analysis. Cell sorting was performed to purify M0 and M2 macrophages. Cell proliferation, collagen synthesis, myofibroblast formation, gene expression of anti-fibrotic and pro-fibrotic factors, MMP-1 activity, and cytokine concentration were investigated. Results showed differentiation of M0 and polarization of M1 and M2 macrophages. M2 macrophages promoted the fibrogenic activities of co-cultured HDF by facilitating cell proliferation, increasing the collagen content, alpha-smooth muscle actin expressed cells, expression of the pro-fibrotic genes and concentration of M2 macrophage related factors, as well as decreasing the expression of the anti-fibrotic genes and MMP-1 activity. These findings reinforce the pro-fibrotic role of M2 macrophages, suggesting therapeutic strategies in fibrotic diseases should target M2 macrophages in the future.
Asunto(s)
Fibrosis/terapia , Activación de Macrófagos , Macrófagos/citología , Macrófagos/inmunología , Metaloproteinasa 1 de la Matriz/metabolismo , Miofibroblastos/metabolismo , Células THP-1/citología , Cicatrización de Heridas/fisiología , Análisis de Varianza , Diferenciación Celular , Proliferación Celular , Técnicas de Cocultivo , Femenino , Fibrosis/patología , HumanosRESUMEN
Objective: Hypertrophic scar (HTS) is a dermal form of fibroproliferative disorder that develops following deep skin injury. HTS can cause deformities, functional disabilities, and aesthetic disfigurements. The pathophysiology of HTS is not understood due to, in part, the lack of an ideal animal model. We hypothesize that human skin with deep dermal wounds grafted onto athymic nude mice will develop a scar similar to HTS. Our aim is to develop a representative animal model of human HTS. Approach: Thirty-six nude mice were grafted with full thickness human skin with deep dermal scratch wound before or 2 weeks after grafting or without scratch. The scratch on the human skin grafts was made using a specially designed jig that creates a wound >0.6 mm in depth. The xenografts were morphologically analyzed by digital photography. Mice were euthanized at 1, 2, and 3 months postoperatively for histology and immunohistochemistry analysis. Results: The mice developed raised and firm scars in the scratched xenografts with more contraction, increased infiltration of macrophage, and myofibroblasts compared to the xenografts without deep dermal scratch wound. Scar thickness and collagen bundle orientation and morphology resembled HTS. The fibrotic scars in the wounded human skin were morphologically and histologically similar to HTS, and human skin epithelial cells persisted in the remodeling tissues for 1 year postengraftment. Innovation and Conclusions: Deep dermal injury in human skin retains its profibrotic nature after transplantation, affording a novel model for the assessment of therapies for the treatment of human fibroproliferative disorders of the skin.
RESUMEN
Hypertrophic scars are caused by trauma or burn injuries to the deep dermis and can cause cosmetic disfigurement and psychological issues. Studies suggest that M2-like macrophages are pro-fibrotic and contribute to hypertrophic scar formation. A previous study from our lab showed that M2 macrophages were present in developing hypertrophic scar tissues in vivo at 3-4 weeks after wounding. In this study, the effect of systemic macrophage depletion on scar formation was explored at subacute phase of wound healing. Thirty-six athymic nude mice that received human skin transplants were randomly divided into macrophage depletion group and control group. The former received intraperitoneal injections of clodronate liposomes while the controls received sterile saline injections on day 7, 10, and 13 postgrafting. Wound area, scar thickness, collagen abundance and collagen bundle structure, mast cell infiltration, myofibroblast formation, M1, and M2 macrophages together with gene expression of M1 and M2 related factors in the grafted skin were investigated at 2, 4, and 8 weeks postgrafting. The transplanted human skin from the control group developed contracted, elevated, and thickened scars while the grafted skin from the depletion group healed with significant less contraction and elevation. Significant reductions in myofibroblast number, collagen synthesis, and hypertrophic fiber morphology as well as mast cell infiltration were observed in the depletion group compared to the control group. Macrophage depletion significantly reduced M1 and M2 macrophage number in the depletion group 2 weeks postgrafting as compared to the control group. These findings suggest that systemic macrophage depletion in subacute phase of wound healing reduces scar formation, which provides evidence for the pro-fibrotic role of macrophages in fibrosis of human skin as well as insight into the potential benefits of specifically depleting M2 macrophages in vivo.
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Cicatriz Hipertrófica/prevención & control , Macrófagos/citología , Macrófagos/fisiología , Cicatrización de Heridas/fisiología , Heridas y Lesiones/terapia , Animales , Diferenciación Celular , Cicatriz Hipertrófica/metabolismo , Cicatriz Hipertrófica/patología , Ácido Clodrónico/farmacología , Colágeno/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibroblastos/citología , Humanos , Liposomas , Macrófagos/efectos de los fármacos , Macrófagos/trasplante , Masculino , Mastocitos/citología , Mastocitos/inmunología , Ratones , Ratones Desnudos , Trasplante de Piel , Bazo/citología , Bazo/inmunología , Cicatrización de Heridas/efectos de los fármacos , Heridas y Lesiones/patologíaRESUMEN
Dermal wound healing, in which transforming growth factor beta 1 (TGFß1) plays an important role, is a complex process. Previous studies suggest that vitamin D has a potential regulatory role in TGFß1 induced activation in bone formation, and there is cross-talk between their signaling pathways, but research on their effects in other types of wound healing is limited. The authors therefore wanted to explore the role of vitamin D and its interaction with low concentration of TGFß1 in dermal fibroblast-mediated wound healing through an in vitro study. Human dermal fibroblasts were treated with vitamin D, TGFß1, both, or vehicle, and then the wound healing functions of dermal fibroblasts were measured. To further explore possible mechanisms explaining the synergistic effect of vitamin D and TGFß1, targeted gene silencing of the vitamin D receptor was performed. Compared to either factor alone, treatment of fibroblasts with both vitamin D and low concentration of TGFß1 increased gene expression of TGFß1, connective tissue growth factor, and fibronectin 1, and enhanced fibroblast migration, myofibroblast formation, and collagen production. Vitamin D receptor gene silencing blocked this synergistic effect of vitamin D and TGFß1 on both collagen production and myofibroblast differentiation. Thus a synergistic effect of vitamin D and low TGFß1 concentration was found in dermal fibroblast-mediated wound healing in vitro. This study suggests that supplementation of vitamin D may be an important step to improve wound healing and regeneration in patients with a vitamin D deficiency.
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Calcitriol/farmacología , Dermis/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Vitaminas/farmacología , Cicatrización de Heridas/efectos de los fármacos , Adulto , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Cromatografía Liquida , Factor de Crecimiento del Tejido Conjuntivo/efectos de los fármacos , Factor de Crecimiento del Tejido Conjuntivo/genética , Dermis/metabolismo , Sinergismo Farmacológico , Femenino , Fibroblastos/metabolismo , Fibronectinas/efectos de los fármacos , Fibronectinas/genética , Humanos , Hidroxiprolina/metabolismo , Técnicas In Vitro , Espectrometría de Masas , Miofibroblastos/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Calcitriol/efectos de los fármacos , Receptores de Calcitriol/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Smad2/efectos de los fármacos , Proteína Smad2/genética , Proteína smad3/efectos de los fármacos , Proteína smad3/genética , Proteína smad7/efectos de los fármacos , Proteína smad7/genética , Factor de Crecimiento Transformador beta1/efectos de los fármacos , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Hypertrophic scars (HTS) are caused by trauma or burn injuries to the deep dermis and are considered fibrosis in the skin. Monocytes, M1 and M2 macrophages are mononuclear phagocytes. Studies suggest that M2 macrophages are profibrotic and might contribute to HTS formation. Our lab has established a human HTS-like nude mouse model, in which the grafted human skin develops red, raised, and firm scarring, resembling HTS seen in humans. In this study, we observed the natural behavior of mononuclear phagocyte system in this nude mouse model of dermal fibrosis at multiple time points. Thirty athymic nude mice received human skin grafts and an equal number of mice received mouse skin grafts as controls. The grafted skin and blood were harvested at 1, 2, 3, 4, and 8 weeks. Wound area, thickness, collagen morphology and level, the cell number of myofibroblasts, M1- and M2-like macrophages in the grafted skin, as well as monocyte fraction in the blood were investigated at each time points. Xenografted mice developed contracted and thickened scars grossly. The xenografted skin resembled human HTS tissue based on enhanced thickness, fibrotic orientation of collagen bundles, increased collagen level, and infiltration of myofibroblasts. In the blood, monocytes dramatically decreased at 1 week postgrafting and gradually returned to normal in the following 8 weeks. In the xenografted skin, M1-like macrophages were found predominantly at 1-2 weeks postgrafting; whereas, M2-like macrophages were abundant at later time points, 3-4 weeks postgrafting coincident with the development of fibrosis in the human skin tissues. This understanding of the natural behavior of mononuclear phagocytes in vivo in our mouse model provides evidence for the role of M2-like macrophages in fibrosis of human skin and suggests that macrophage depletion in the subacute phases of wound healing might reduce or prevent HTS formation.
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Cicatriz Hipertrófica/inmunología , Citocinas/genética , Macrófagos/inmunología , Monocitos/inmunología , Miofibroblastos/citología , ARN Mensajero/metabolismo , Trasplante de Piel , Piel/inmunología , Cicatrización de Heridas/inmunología , Animales , Cicatriz Hipertrófica/patología , Colágeno , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Fibrosis , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Hidroxiprolina/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Desnudos , Sistema Mononuclear Fagocítico/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/patología , Trasplante HeterólogoRESUMEN
Hypertrophic scars (HTS) are generally believed to result from proliferation and activation of resident connective tissue fibroblasts after burns. To demonstrate a potential role of blood-borne cells, the peripheral blood mononuclear cells (PBMCs) and the effect of PBMCs on dermal fibroblast behavior was investigated. Flow cytometry was used to analyze the surface and intracellular protein expression of PBMCs and fibroblasts. Transwell migration assay, enzyme-linked immunosorbent assay and real-time reverse transcription polymerase chain reaction was performed to assess fibroblast functions. We identified a novel subpopulation of PBMCs in burn patients in vivo that appears at an early stage following major thermal injuries, which primarily express procollagen 1, leukocyte specific protein 1, CD204, toll-like receptor 4 and stromal cell-derived factor 1 (SDF-1) receptor CXCR4. In vitro, the conditioned media from burn patient PBMCs up-regulated the expression of fibrotic growth factors and extracellular matrix molecules, down-regulated antifibrotic factor decorin, enhanced cell chemotaxis and promoted cell differentiation into contractile myofibroblasts in dermal fibroblasts. After thermal injury, this novel subpopulation of PBMCs is systemically triggered and attracted to the wounds under SDF-1/CXCR4 signaling where they appear to modulate the functions of resident connective tissue cells and thus contribute to the development of HTS.
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Quemaduras/sangre , Quimiotaxis , Cicatriz Hipertrófica/sangre , Leucocitos Mononucleares/citología , Macrófagos/citología , Adulto , Superficie Corporal , Quemaduras/patología , Diferenciación Celular , Ensayos de Migración Celular , Células Cultivadas , Células del Tejido Conectivo/citología , Células del Tejido Conectivo/metabolismo , Decorina/metabolismo , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibroblastos/citología , Humanos , Técnicas In Vitro , Leucocitos Mononucleares/metabolismo , Macrófagos/metabolismo , Masculino , Proteínas de Microfilamentos/metabolismo , Persona de Mediana Edad , Miofibroblastos/citología , Procolágeno/metabolismo , Receptores CXCR4/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptores Depuradores de Clase A/metabolismo , Transducción de Señal , Piel/citología , Receptor Toll-Like 4/metabolismo , Índices de Gravedad del Trauma , Adulto JovenRESUMEN
Heterotopic ossification (HO) is a complication of musculoskeletal injury characterized by the formation of mature bone in soft tissues. The etiology of HO is unknown. We investigated the role of bone marrow derived progenitor cells in HO pathophysiology. We isolated the cells from HO specimens by cell explantation. Using flow cytometry and immunofluorescence microscopy, we found that 35 to 65% of the HO cells exhibit a bone marrow derived fibrocyte profile consisting in spindle-shaped morphology associated with type 1 pro-collagen and LSP1 expression. When cultured in osteogenic differentiation medium, active machinery for bone mineralization (high gene expression of Anx2, TNAP, and Pit-1), and calcium/phosphate deposits were found. Interestingly, interferon-alpha 2b significantly reduced the proliferation rate and COL1 gene expression in HO cells. We have characterized a novel subset of bone marrow derived progenitor cells in the HO specimens. The findings from this research study will provide new insights into the development of HO in burn patients.
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Células Progenitoras Endoteliales/metabolismo , Osificación Heterotópica/patología , Osteogénesis , Animales , Quemaduras/metabolismo , Inmunohistoquímica , Microscopía Electrónica , Células Madre/metabolismoRESUMEN
Effective prevention and treatment of hypertrophic scars (HTSs), a dermal form of fibrosis that frequently occurs following thermal injury to deep dermis, are unsolved significant clinical problems. Previously, we have found that stromal cell-derived factor 1/CXCR4 signaling is up-regulated during wound healing in burn patients and HTS tissue after thermal injury. We hypothesize that blood-borne mononuclear cells are recruited into wound sites after burn injury through the chemokine pathway of stromal cell-derived factor 1 and its receptor CXCR4. Deep dermal injuries to the skin are often accompanied by prolonged inflammation, which leads to chemotaxis of mononuclear cells into the wounds by chemokine signaling where fibroblast activation occurs and ultimately HTS are formed. Blocking mononuclear cell recruitment and fibroblast activation, CXCR4 antagonism is expected to reduce or minimize scar formation. In this study, the inhibitory effect of CXCR4 antagonist CTCE-9908 on dermal fibrosis was determined in vivo using a human HTS-like nude mouse model, in which split-thickness human skin is transplanted into full-thickness dorsal excisional wounds in athymic mice, where these wounds subsequently develop fibrotic scars that resemble human HTS as previously described. CTCE-9908 significantly attenuated scar formation and contraction, reduced the accumulation of macrophages and myofibroblasts, enhanced the remodeling of collagen fibers, and down-regulated the gene and protein expression of fibrotic growth factors in the human skin tissues. These findings support the potential therapeutic value of CXCR4 antagonist in dermal fibrosis and possibly other fibroproliferative disorders.
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Cicatriz Hipertrófica/prevención & control , Dermis/efectos de los fármacos , Péptidos/farmacología , Receptores CXCR4/antagonistas & inhibidores , Cicatrización de Heridas/efectos de los fármacos , Adulto , Animales , Cicatriz Hipertrófica/patología , Dermis/patología , Modelos Animales de Enfermedad , Femenino , Fibrosis , Humanos , Masculino , Ratones Desnudos , Persona de Mediana Edad , Trasplante de PielRESUMEN
Hypertrophic scars are a significant fibroproliferative disorder complicating deep injuries to the skin. We hypothesize that activated deep dermal fibroblasts are subject to regulation by bone marrow-derived mesenchymal stem cells (BM-MSCs), which leads to the development of excessive fibrosis following deep dermal injury. We found that the expression of fibrotic factors was higher in deep burn wounds compared with superficial burn wounds collected from burn patients with varying depth of skin injury. We characterized deep and superficial dermal fibroblasts, which were cultured from the deep and superficial dermal layers of normal uninjured skin obtained from abdominoplasty patients, and examined the paracrine effects of BM-MSCs on the fibrotic activities of the cells. In vitro, deep dermal fibroblasts were found higher in the messenger RNA (mRNA) levels of type 1 collagen, alpha smooth muscle actin, transforming growth factor beta, stromal cell-derived factor 1, and tissue inhibitor of metalloproteinase 1, an inhibitor of collagenase (matrix metalloproteinase 1). As well, deep dermal fibroblasts had low matrix metalloproteinase 1 mRNA, produced more collagen, and contracted collagen lattices significantly greater than superficial fibroblasts. By co-culturing layered fibroblasts with BM-MSCs in a transwell insert system, BM-MSCs enhanced the fibrotic behavior of deep dermal fibroblasts, which suggests a possible involvement of BM-MSCs in the pathogenesis of hypertrophic scarring.
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Quemaduras/patología , Cicatriz Hipertrófica/prevención & control , Piel/patología , Trasplante de Células Madre/métodos , Cicatrización de Heridas/fisiología , Adulto , Quemaduras/metabolismo , Quemaduras/cirugía , Proliferación Celular , Células Cultivadas , Cicatriz Hipertrófica/metabolismo , Cicatriz Hipertrófica/patología , Colágeno/biosíntesis , Colágeno/genética , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica , Humanos , Flujometría por Láser-Doppler , Masculino , Células Madre Mesenquimatosas , Persona de Mediana Edad , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Piel/metabolismoRESUMEN
The purpose of this study was to develop polymeric nano-carriers of doxorubicin (DOX) that can increase the therapeutic efficacy of DOX for sensitive and resistant cancers. Towards this goal, two polymeric DOX nano-conjugates were developed, for which the design was based on the use of multi-functionalized poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) micelles decorated with alphavbeta3 integrin-targeting ligand (i.e. RGD4C) on the micellar surface. In the first formulation, DOX was conjugated to the degradable PEO-b-PCL core using the pH-sensitive hydrazone bonds, namely RGD4C-PEO-b-P(CL-Hyd-DOX). In the second formulation, DOX was conjugated to the core using the more stable amide bonds, namely RGD4C-PEO-b-P(CL-Ami-DOX). The pH-triggered drug release, cellular uptake, intracellular distribution, and cytotoxicity against MDA-435/LCC6(WT) (a DOX-sensitive cancer cell line) and MDA-435/LCC6(MDR) (a DOX-resistant clone expressing a high level of P-glycoprotein) were evaluated. Following earlier in vitro results, SCID mice bearing MDA-435/LCC6(WT) and MDA-435/LCC6(MDR) tumors were treated with RGD4C-PEO-b-P(CL-Hyd-DOX) and RGD4C-PEO-b-P(CL-Ami-DOX), respectively. In both formulations, surface decoration with RGD4C significantly increased the cellular uptake of DOX in MDA-435/LCC6(WT) and MDA-435/LCC6(MDR) cells. In MDA-435/LCC6(WT), the best cytotoxic response was achieved using RGD4C-PEO-b-P(CL-Hyd-DOX), that correlated with the highest cellular uptake and preferential nuclear accumulation of DOX. In MDA-435/LCC6(MDR), RGD4C-PEO-b-P(CL-Ami-DOX) was the most cytotoxic, and this effect correlated with the accumulation of DOX in the mitochondria. Studies using a xenograft mouse model yielded results parallel to those of the in vitro studies. Our study showed that RGD4C-decorated PEO-b-P(CL-Hyd-DOX) and PEO-b-P(CL-Ami-DOX) can effectively improve the therapeutic efficacy of DOX in human MDA-435/LCC6 sensitive and resistant cancer, respectively, pointing to the potential of these polymeric micelles as the custom-designed drug carriers for clinical cancer therapy.
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Antibióticos Antineoplásicos/uso terapéutico , Doxorrubicina/química , Doxorrubicina/uso terapéutico , Sistemas de Liberación de Medicamentos/métodos , Nanoestructuras/química , Poliésteres/química , Polímeros/química , Animales , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Humanos , Ratones , Micelas , Modelos Teóricos , Neoplasias/tratamiento farmacológico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The efficiency of cancer immunotherapy strategies is hampered by the existence of an intra-tumoral immunosuppressive environment involving tolerogenic dendritic cells (DCs) and regulatory T (T(reg)) cells. Hyperactivation of STAT3 in tumor is implicated in the generation of this immunosuppressive environment. The purpose of this study was to test whether simultaneous inhibition of STAT3 in tumor and TLR4 ligand-induced activation of DCs can modulate tumor-induced immunosuppression. For this purpose, the effects of a TLR4 ligand, 7-acyl lipid A, delivered by poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs) to DCs on the activity of DCs and T(reg) cells was evaluated in vitro. In addition the immunomodulatory and anticancer effects of 7-acyl lipid A PLGA-NPs in combination with a STAT3 inhibitory agent, JSI-124, in a B16 mouse melanoma model was explored, in vivo. PLGA-NP delivery of 7-acyl lipid A to DCs reduced the suppressive effects of T(reg) cells on T cells in vitro. Besides, daily Intra-tumoral co-administration of 7-acyl lipid A PLGA-NPs and JSI-124 in C57BL/6 mice bearing B16-F10 tumor for 8 days resulted in a significant increase in the percentage of tumor infiltrated T cells as compared with control group that received PBS and monotherapy groups. The average tumor volume in the tumor-bearing mice that received JSI-124 plus 7-acyl lipid A PLGA-NPs combination therapy was found to be significantly lower than that in PBS and monotherapy groups. Our findings show a potential for the combination of STAT3 inhibition in tumor and TLR4 induced DC activation in increasing the efficacy of cancer immunotherapy.
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Adyuvantes Inmunológicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Sistemas de Liberación de Medicamentos , Tolerancia Inmunológica/efectos de los fármacos , Lípido A/análogos & derivados , Melanoma Experimental/tratamiento farmacológico , Factor de Transcripción STAT3/antagonistas & inhibidores , Triterpenos/administración & dosificación , Animales , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Glicolatos/administración & dosificación , Tolerancia Inmunológica/inmunología , Inyecciones Intralesiones , Ácido Láctico , Lípido A/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
The purpose of this study was to evaluate the efficacy of poly(lactic-co-glycolic acid) (PLGA)-based vaccines in breaking immunotolerance to cancer-associated self-antigens. Vaccination of mice bearing melanoma B16 tumors with PLGA nanoparticles (NP) co-encapsulating the poorly immunogenic melanoma antigen, tyrosinase-related protein 2 (TRP2), along with Toll-like receptor (TLR) ligand (7-acyl lipid A) was examined. Remarkably, this vaccine was able to induce therapeutic anti-tumor effect. Activated TRP2-specific CD8 T cells were capable of interferon (IFN)-gamma secretion at lymph nodes and spleens of the vaccinated mice. More importantly, TRP2/7-acyl lipid A-NP treated group has shown immunostimulatory milieu at the tumor microenvironment, as evidenced by increased level of pro-inflammatory cytokines compared to control group. These results support the potential use of PLGA nanoparticles as competent carriers for future cancer vaccine formulations.
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Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Ácido Láctico/uso terapéutico , Melanoma Experimental/terapia , Ácido Poliglicólico/uso terapéutico , Receptor Toll-Like 4/inmunología , Animales , Células Cultivadas , Citotoxicidad Inmunológica/inmunología , Portadores de Fármacos/uso terapéutico , Interferón gamma/inmunología , Oxidorreductasas Intramoleculares/inmunología , Ácido Láctico/administración & dosificación , Lípido A/inmunología , Ratones , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , Nanopartículas/uso terapéutico , Ácido Poliglicólico/administración & dosificación , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
One of the major limitations for cancer immunotherapy is related to the frequent existence of an intra-tumoral immunosuppressive environment, to which STAT3 (Signal transducer and activator of transcription-3) activation in tumor and dendritic cells (DCs) are believed to contribute. In this study, we tested the hypothesis that the combination of CpG (a DC activator) and JSI-124 (a STAT3 inhibitor) may generate synergistic antitumor effects compared to CpG or JSI-124 alone. B16-F10, a mouse melanoma cell line that has constitutively active STAT3, was grafted in C57BL/6 mice and then tumor-bearing mice treated intra-tumorally with (a) phosphate buffered saline, (b) 10 microg CpG, (c) 1 mg kg(-1) JSI-124 or (d) 10 microg CpG+1 mg kg(-1) JSI-124. The effects of treatments on tumor growth, survival and antitumor immune responses were evaluated. Although significant antitumor effects were detected with the single-agent treatments, the CpG+JSI-124 treatment resulted in synergistic antitumor effects compared to CpG or JSI-124 alone. Correlating with these findings, the combination therapy resulted in significantly higher intra-tumoral levels of several proinflammatory, TH1-related cytokines (including IL-12, IFN-gamma, TNF-alpha and IL-2), increases in intra-tumoral CD8+ and CD4+ T cells expressing activation/memory markers and NK cells and increases in activated DCs in the tumors and regional lymph nodes (LNs). Concomitantly, the combination therapy led to a significantly decreased level of immunosuppression, as evidenced by lower intra-tumoral level of VEGF and TGF-beta, and decreased number of CD4+CD25+Foxp3+ regulatory T cells in the regional LNs. This study has provided the proof-of-principle for combining CpG and JSI-124 to enhance antitumor immune responses.
Asunto(s)
Sinergismo Farmacológico , Melanoma Experimental/terapia , Oligodesoxirribonucleótidos/uso terapéutico , Factor de Transcripción STAT3/antagonistas & inhibidores , Triterpenos/uso terapéutico , Animales , Western Blotting , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Línea Celular Tumoral , Terapia Combinada , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Interleucina-2/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Masculino , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
PURPOSE: Resveratrol is a well-known chemopreventive and chemotherapeutic agent. Among all of the resveratrol analogs synthesized, 3,4,5,4'-tetramethoxystilbene (DMU-212) shows high activity and selectivity against various cancer cell types. The objective of this study is to investigate why DMU-212 has higher anti-tumor activity than resveratrol. METHODS: The effects of DMU-212 and resveratrol on cell viability, cell cycle, Stat3 activation, and microtubule dynamic were investigated and compared using MTT assay, cell cycle analysis, Western blot, tubulin polymerization assay, respectively, in MDA-MB-435 and MCF-7 human breast cancer cells. RESULTS: Compared to resveratrol, DMU-212 exerted a significantly higher growth inhibition in both cell lines. Further studies demonstrated that DMU-212 acted via different mechanisms from resveratrol. First, DMU-212 induced predominantly G2/M arrest whereas resveratrol induced G0/G1 arrest in both cell lines. Correlating with these findings, resveratrol induced more dramatic changes in the expression of Cyclin D1 compared to DMU-212. Second, DMU-212 induced apoptosis and reduced the expression of multiple anti-apoptotic proteins more appreciably than resveratrol. Third, while both agents inhibited Stat3 phosphorylation, treatments of DMU-212 but not resveratrol led to a significant increase in tubulin polymerization. The higher sensitivity to DMU-122 in MDA-MB-435 correlated with the more prominent effects seen in these parameters in this cell line, as compared to MCF7. CONCLUSION: Compared to resveratrol, the novel stilbene derivative, DMU-212, had higher anti-tumor effects, which are likely owing to its modulation of multiple cellular targets.
Asunto(s)
Adenocarcinoma/patología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Estilbenos/uso terapéutico , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/genética , Biopolímeros/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Estrógenos , Femenino , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Neoplasias Hormono-Dependientes/patología , Resveratrol , Factor de Transcripción STAT3/antagonistas & inhibidores , Estilbenos/farmacología , Tubulina (Proteína)/metabolismoRESUMEN
Poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) and newly developed poly(ethylene oxide)-block-poly(alpha-benzyl carboxylate epsilon-caprolactone) (PEO-b-PBCL) micelles were evaluated for the solubilization and delivery of cucurbitacin I and B, poorly water soluble inhibitors of signal transducer and activator of transcription 3 (STAT3). Encapsulation of cucurbitacins in PEO-b-PCL and PEO-b-PBCL by co-solvent evaporation technique resulted in polymeric micelles <90 nm in diameter. The aqueous solubility of both derivatives increased from less than 0.05 mg/mL in the absence of the copolymer to around 0.30-0.44 and 0.65-0.68 mg/mL in the presence of 5000-5000 and 5000-24,000 PEO-b-PCL micelles, respectively. Maximum cucurbitacin solubilization was achieved with PEO-b-PBCL micelles for both derivatives. PEO-b-PCL micelles having longer PCL block were found to be more efficient in sustaining the rate of release for cucurbitacins. The anti-cancer and STAT3 inhibitory activity of polymeric micellar cucurbitacins were comparable with free drugs in B16.F10 melanoma cell line in vitro. Intratumoral injection of 1 mg/kg/day cucurbitacin I resulted in the regression of established B16.F10 mouse melanoma tumors in vivo. In comparison to free cucurbitacin I, PEO-b-PBCL micellar cucurbitacin I was found to provide comparable anti-cancer effects against B16.F10 tumors and limit drug levels in animal serum while maintaining high drug levels in tumor following intratumoral administration. The results indicate the potential of polymeric micelles as suitable vehicles for the delivery of cucurbitacin- I and B.
Asunto(s)
Cucurbitacinas/uso terapéutico , Melanoma Experimental/tratamiento farmacológico , Micelas , Polímeros/química , Factor de Transcripción STAT3/antagonistas & inhibidores , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Derivados del Benceno/química , Disponibilidad Biológica , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cucurbitacinas/administración & dosificación , Cucurbitacinas/farmacocinética , Sistemas de Liberación de Medicamentos/métodos , Concentración 50 Inhibidora , Lactonas/química , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Poliésteres/química , Polietilenglicoles/química , Factor de Transcripción STAT3/metabolismo , Solubilidad , Triterpenos/administración & dosificación , Triterpenos/farmacocinética , Triterpenos/uso terapéuticoRESUMEN
Curcumin is recognized as a potential chemotherapeutic agent against a variety of tumors. However, the clinical application of curcumin is hindered due to its poor water solubility and fast degradation. The objective of this study was to investigate amphiphilic block copolymer micelles of poly(ethylene oxide)-b-poly(epsilon-caprolactone) (PEO-PCL) as vehicles for the solubilization, stabilization, and controlled delivery of curcumin. Curcumin-loaded PEO-PCL micelles were prepared by a cosolvent evaporation technique. PEO-PCL micelles were able to solubilize curcumin effectively, protect the encapsulated curcumin from hydrolytical degradation in physiological matrix, and control the release of curcumin over a few days. The characteristics of resultant micelles were found to depend on the polymerization degrees of epsilon-caprolactone. Among different PEO-PCL micelles, PEO(5000)-PCL(24500) was the most efficient in solubilizing curcumin while PEO(5000)-PCL(13000) was the best carrier in reducing its release rate. PEO-PCL micelle-encapsulated curcumin retained its cytotoxicity in B16-F10, a mouse melanoma cell line, and SP-53, Mino, and JeKo-1 human mantle cell lymphoma cell lines. These results demonstrated the potential of PEO-PCL micelles as an injectable formulation for efficient solubilization, stabilization, and controlled delivery of curcumin.
Asunto(s)
Curcumina/administración & dosificación , Preparaciones de Acción Retardada/química , Lactonas/uso terapéutico , Polietilenglicoles/uso terapéutico , Animales , Antineoplásicos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Curcumina/farmacología , Estabilidad de Medicamentos , Humanos , Ratones , Micelas , SolubilidadRESUMEN
A simple, rapid and reliable high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of curcumin in rat plasma. Plasma was precipitated with acetonitrile after addition of the internal standard (IS), 4-hydroxybenzophenone. Separation was achieved on a Waters muBondapak C(18) column (3.9 x 300 mm, 5 microm) using acetonitrile (55%) and citric buffer, pH 3.0 (45%) as the mobile phase (flow rate = 1.0 mL/min). The UV detection wavelength was 300 and 428 nm for IS and curcumin, respectively. The extraction efficiencies were 97.08, 95.69 and 94.90% for 50, 200 and 1000 ng/mL of curcumin in rat plasma, respectively. The calibration curve was linear over the range 0.02-1 microg/mL with a correlation coefficient of r(2) > 0.999. The intra- and inter-day coefficients of variation were less than 13%, and mean intra- and inter-day errors were less than +/-6% at 50, 200 and 1000 ng/mL of curcumin. This assay was successfully applied to the pharmacokinetic studies of both solubilized curcumin and its polymeric micellar formulation in rats. It was found that polymeric micelles increased the half-life of curcumin 162-fold that of solubilized curcumin and increased the volume of distribution (Vd(ss)) by 70-fold.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Curcumina/farmacocinética , Polímeros/química , Animales , Micelas , Ratas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrofotometría UltravioletaRESUMEN
The objective of the present study was to evaluate the effects of formulation parameters on the in vivo pharmacological activity of the chitosan-insulin nanoparticles. Chitosan-insulin nanoparticles were prepared by ionotropic gelation at pH 5.3 and 6.1 and denoted as F5.3 np and F6.1 np, respectively. F5.3 np and F6.1 np administered orally at insulin doses of 50 U/kg and/or 100 U/kg were effective at lowering the serum glucose level of streptozotocin-induced diabetic rats. The 100 U/kg-dose F5.3 np sustained the serum glucose at pre-diabetic levels for at least 11 h. In comparison, F6.1 np had a faster onset of action (2h versus 10h) but lower efficiency. The effectiveness of peroral F5.3 np and F6.1 np in lowering the serum glucose level of streptozotocin-induced diabetic rats was ascribed to the local effect of insulin in intestine. Confocal micrographs showed strong interaction between rat intestinal epithelium and chitosan nanoparticles 3h post-oral administration.
Asunto(s)
Quitosano/farmacología , Diabetes Mellitus Experimental/sangre , Insulina/farmacología , Nanoestructuras , Administración Oral , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Quitosano/administración & dosificación , Diabetes Mellitus Experimental/tratamiento farmacológico , Insulina/administración & dosificación , Masculino , Ratas , Ratas WistarRESUMEN
Benign prostate hyperplasia and prostate cancer are major public health problems. We report herein that daily treatment of male rats with 50, 100 or 150 mg quercetin per kg body weight resulted in serum concentrations of quercetin equivalent to 25.3 microM, 43.3 microM and 54.3 microM respectively. Concomitantly, serum testosterone levels were increased by 1.79-, 1.83- and 3.48-fold, while serum dihydrotestosterone (DHT) levels were 125%, 92% and 73% of the control. A slight increase in prostate weight coupled with dilated prostate lumens full of secretory materials were observed. Finasteride alone caused a significant decrease in serum DHT level and prostate weight. Co-administration of quercetin with finasteride prevented the finasteride-induced decrease in serum DHT levels but significantly enhanced the reduction in wet prostate weight, which was reduced by 26.9% in finasteride-treated animals to 31.8%, 40.0% and 48.2% after finasteride given together with the three doses of quercetin. The combined treatment altered cell cycle-regulated proteins in a wide spectrum. The expressions of cyclin D1, CDK-4, cdc-2 and phospho-cdc-2 at tyrosine 15, phospho-MEK1/2, phospho-MAP kinase, phospho-pRb at serine 780 and serine 807/811 were significantly inhibited, while the levels of p15, p21 and p27 were increased. In conclusion, quercetin-finasteride treatments caused wide cell cycle deregulation in rat prostates, which, in turn, decreased the proliferation rate, changed the secretion activities of epithelial cells and resulted in a marked reduction in wet prostate weight. The results suggest that quercetin synergizes with finasteride to reduce the wet prostate weight through a cell cycle-related pathway, which may be androgen independent.