RESUMEN
BACKGROUND: Tumour associated macrophages (TAMs) are important players in breast tumour progression and metastasis. Clinical and preclinical evidence suggests a role for zoledronate (ZOL) in breast cancer metastasis prevention. Further, zoledronate is able to induce inflammatory activation of monocytes and macrophages, which can be favourable in cancer treatments. The inherent bone tropism of zoledronate limits its availability in soft tissues and tumours. In this study we utilised an orthotopic murine breast cancer model to evaluate the possibility to use liposomes (EMP-LIP) to target zoledronate to tumours to modify TAM activation. METHODS: Triple-negative breast cancer 4T1 cells were inoculated in the 4th mammary fat pad of female Balb/c mice. Animals were divided according to the treatment: vehicle, ZOL, EMP-LIP and liposome encapsulated zoledronate (ZOL-LIP). Treatment was done intravenously (with tumour resection) and intraperitoneally (without tumour resection). Tumour growth was followed by bioluminescence in vivo imaging (IVIS) and calliper measurements. Tumour-infiltrating macrophages were assessed by immunohistochemical and immunofluorescence staining. Protein and RNA expression levels of inflammatory transcription factors and cytokines were measured by Western Blotting and Taqman RT-qPCR. RESULTS: Liposome encapsulated zoledronate (ZOL-LIP) treatment suppressed migration of 4T1 cell in vitro. Tumour growth and expression of the angiogenic marker CD34 were reduced upon both ZOL and ZOL-LIP treatment in vivo. Long-term ZOL-LIP treatment resulted in shift towards M1-type macrophage polarization, increased CD4 T cell infiltration and activation of NF-κB indicating changes in intratumoural inflammation, whereas ZOL treatment showed similar but non-significant trends. Moreover, ZOL-LIP had a lower bisphosphonate accumulation in bone compared to free ZOL. CONCLUSION: Results show that the decreased bisphosphonate accumulation in bone promotes the systemic anti-tumour effect of ZOL-LIP by increasing inflammatory response in TNBC tumours via M1-type macrophage activation.
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Liposomas , Neoplasias de la Mama Triple Negativas , Humanos , Femenino , Ratones , Animales , Ácido Zoledrónico/farmacología , Liposomas/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Línea Celular Tumoral , Difosfonatos/uso terapéutico , Difosfonatos/farmacología , Macrófagos , Ratones Endogámicos BALB CRESUMEN
Bisphosphonates (BP) are a class of calcium-binding drug used to prevent bone resorption in skeletal disorders such as osteoporosis and metastatic bone disease. They act by selectively targeting bone-resorbing osteoclasts and can be grouped into two classes depending on their intracellular mechanisms of action. Simple BPs cause osteoclast apoptosis after cytoplasmic conversion into toxic ATP analogues. In contrast, nitrogen-containing BPs potently inhibit FPP synthase, an enzyme of the mevalonate (cholesterol biosynthesis) pathway. This results in production of a toxic metabolite (ApppI) and the loss of long-chain isoprenoid lipids required for protein prenylation, a process necessary for the function of small GTPase proteins essential for the survival and activity of osteoclasts. In this review we provide a state-of-the-art overview of these mechanisms of action and a historical perspective of how they were discovered. Finally, we challenge the long-held dogma that BPs act only in the skeleton and highlight recent studies that reveal insights into hitherto unknown effects on tumour-associated and tissue-resident macrophages.
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Resorción Ósea , Difosfonatos , Resorción Ósea/tratamiento farmacológico , Huesos , Difosfonatos/farmacología , Humanos , Osteoclastos , Prenilación de ProteínaRESUMEN
Pain is the most unbearable symptom accompanying primary bone cancers and bone metastases. Bone resorptive disorders are often associated with hypercalcemia, contributing to the pathologic process. Nitrogen-containing bisphosphonates (NBPs) are efficiently used to treat bone cancers and metastases. Apart from their toxic effect on cancer cells, NBPs also provide analgesia via poorly understood mechanisms. We previously showed that NBPs, by inhibiting the mevalonate pathway, induced formation of novel ATP analogs such as ApppI [1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) triphosphoric acid diester], which can potentially be involved in NBP analgesia. In this study, we used the patch-clamp technique to explore the action of ApppI on native ATP-gated P2X receptors in rat sensory neurons and rat and human P2X3, P2X2, and P2X7 receptors expressed in human embryonic kidney cells. We found that although ApppI has weak agonist activity, it is a potent inhibitor of P2X3 receptors operating in the nanomolar range. The inhibitory action of ApppI was completely blocked in hypercalcemia-like conditions and was stronger in human than in rat P2X3 receptors. In contrast, P2X2 and P2X7 receptors were insensitive to ApppI, suggesting a high selectivity of ApppI for the P2X3 receptor subtype. NBP, metabolite isopentenyl pyrophosphate, and endogenous AMP did not exert any inhibitory action, indicating that only intact ApppI has inhibitory activity. Ca2+-dependent inhibition was stronger in trigeminal neurons preferentially expressing desensitizing P2X3 subunits than in nodose ganglia neurons, which also express nondesensitizing P2X2 subunits. Altogether, we characterized previously unknown purinergic mechanisms of NBP-induced metabolites and suggest ApppI as the endogenous pain inhibitor contributing to cancer treatment with NBPs.
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Adenosina Trifosfato/análogos & derivados , Calcio/farmacología , Activación del Canal Iónico/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X3 , Adenosina Trifosfato/farmacología , Animales , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Activación del Canal Iónico/fisiología , Masculino , Ratas , Ratas Wistar , Receptores Purinérgicos P2X3/fisiologíaRESUMEN
PURPOSE: We have examined the uptake routes by which breast cancer cells internalize different formulations of nitrogen containing bisphosphonates (N-BPs). METHODS: Cell viability was assessed with the tetrazolium colorimetric test (MTT assay) after treatment with different N-BP formulations in the presence or absence of inhibitors for different endocytosis mechanisms. Intracellular formation of isopentenyl pyrophosphate (IPP) and triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (ApppI), were quantified with mass spectrometry (ES-LTQ-MS) as surrogate markers for N-BP efficacy. Direct quantification intracellular [(14)C]-labeled zoledronic acid was done with liquid scintillation counting. RESULTS: The main uptake route for all the different formulations of nitrogen containing bisphosphonates was shown to be dynamin dependent endocytosis, which was significantly enhanced with calcium. This uptake mechanism was mostly caveolin and clathrin independent in MCF7 cells, but more clathrin dependent in T47D cells. Liposome encapsulation of the drug shifted the uptake mechanism to be more dependent on caveolin in both the cell lines. The cytotoxicity of N-BPs and the concentrations of formed intracellular ApppI and IPP were significantly increased by calcium chelation and liposome encapsulation, the latter being the most potent formulation. CONCLUSION: Nitrogen containing bisphosphonates require active endocytosis for cellular uptake and in the breast cancer cells the mechanism is uniformly dynamin dependent for all the formulations tested. This differs e.g. from the previous observations on macrophages, which mostly utilize macropinocytosis. Liposomal formulation was found to prolong the duration of the drug effect in cells.
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Neoplasias de la Mama/metabolismo , Difosfonatos/administración & dosificación , Imidazoles/administración & dosificación , Ácido Risedrónico/administración & dosificación , Calcio/química , Línea Celular Tumoral , Difosfonatos/química , Difosfonatos/farmacología , Endocitosis , Femenino , Humanos , Imidazoles/química , Imidazoles/farmacología , Liposomas , Nitrógeno/química , Ácido Risedrónico/química , Ácido Risedrónico/farmacología , Ácido ZoledrónicoRESUMEN
INTRODUCTION: The immune system plays a major role in cancer progression. In solid tumors, 5-40 % of the tumor mass consists of tumor-associated macrophages (TAMs) and there is usually a correlation between the number of TAMs and poor prognosis, depending on the tumor type. TAMs usually resemble M2 macrophages. Unlike M1-macrophages which have pro-inflammatory and anti-cancer functions, M2-macrophages are immunosuppressive, contribute to the matrix-remodeling, and hence favor tumor growth. The role of TAMs is not fully understood in breast cancer progression. METHODS: Macrophage infiltration (CD68) and activation status (HLA-DRIIα, CD163) were evaluated in a large cohort of human primary breast tumors (562 tissue microarray samples), by immunohistochemistry and scored by automated image analysis algorithms. Survival between groups was compared using the Kaplan-Meier life-table method and a Cox multivariate proportional hazards model. Macrophage education by breast cancer cells was assessed by ex vivo differentiation of peripheral blood mononuclear cells (PBMCs) in the presence or absence of breast cancer cell conditioned media (MDA-MB231, MCF-7 or T47D cell lines) and M1 or M2 inducing cytokines (respectively IFN-γ, IL-4 and IL-10). Obtained macrophages were analyzed by flow cytometry (CD14, CD16, CD64, CD86, CD200R and CD163), ELISA (IL-6, IL-8, IL-10, monocyte colony stimulating factor M-CSF) and zymography (matrix metalloproteinase 9, MMP-9). RESULTS: Clinically, we found that high numbers of CD163(+) M2-macrophages were strongly associated with fast proliferation, poor differentiation, estrogen receptor negativity and histological ductal type (p<0.001) in the studied cohort of human primary breast tumors. We demonstrated ex vivo that breast cancer cell-secreted factors modulate macrophage differentiation toward the M2 phenotype. Furthermore, the more aggressive mesenchymal-like cell line MDA-MB231, which secretes high levels of M-CSF, skews macrophages toward the more immunosuppressive M2c subtype. CONCLUSIONS: This study demonstrates that human breast cancer cells influence macrophage differentiation and that TAM differentiation status correlates with recurrence free survival, thus further emphasizing that TAMs can similarly affect therapy efficacy and patient outcome.
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Neoplasias de la Mama/patología , Leucocitos Mononucleares/patología , Macrófagos/patología , Neoplasias de la Mama/metabolismo , Diferenciación Celular/fisiología , Línea Celular Tumoral , Proliferación Celular/fisiología , Citocinas/metabolismo , Femenino , Humanos , Leucocitos Mononucleares/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismoRESUMEN
BACKGROUND: Tumour stromal macrophages differentiate to tumour-associated macrophages (TAMs) with characteristics of immunosuppressive M2-type macrophages, having a central role in promoting tumour vascularisation, cancer cell dissemination and in suppressing anti-cancer immune responses. Bisphosphonates (BPs) are a group of drugs commonly used as anti-resorptive agents. Further, nitrogen containing BPs like Zoledronate (ZOL), are known to cause unspecific inflammatory reactions hence the hypothesis that its use could modulate TAMs polarization toward a more inflammatory phenotype. METHODS: We studied the in vitro polarization of J774 murine macrophages upon culture in 4T1 breast cancer cell-conditioned medium (4T1CM) and stimulation with LPS and free and liposome-encapsulated bisphosphonates. RESULTS: In this system, breast cancer soluble factors reduced the pro-inflammatory activation of macrophages but increased the secretion of matrix metalloproteinases (MMPs). In the presence of 4T1CM, a non-cytotoxic dose of liposome-encapsulated ZOL (ZOL-LIP) enhanced the expression of iNOS and TNF-α, markers of M1 activation, but did not diminish the expression of M2-type markers. In contrast, clodronate treatment either as a free drug (CLO) or liposome-encapsulated (CLO-LIP) decreased the expression of the M1-type markers and was highly cytotoxic to the macrophages. CONCLUSIONS: Breast cancer cells soluble factors modulate macrophages toward M2 activation state. Bisphosphonates may be applied to counteract this modulation. We propose that ZOL-LIP may be suitable for favouring cytotoxic immune responses by TAMs in breast cancer, whereas CLO-LIP may be appropriate for TAM depletion.
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Neoplasias de la Mama/metabolismo , Medios de Cultivo Condicionados/farmacología , Difosfonatos/farmacología , Imidazoles/farmacología , Macrófagos/efectos de los fármacos , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Liposomas , Macrófagos/citología , Macrófagos/inmunología , Metaloproteinasas de la Matriz/metabolismo , Ratones , Óxido Nítrico Sintasa de Tipo II/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ácido ZoledrónicoRESUMEN
Most human γδ T cells express Vγ2Vδ2 TCRs and play important roles in microbial and tumor immunity. Vγ2Vδ2 T cells are stimulated by self- and foreign prenyl pyrophosphate intermediates in isoprenoid synthesis. However, little is known about the molecular basis for this stimulation. We find that a mAb specific for butyrophilin 3 (BTN3)/CD277 Ig superfamily proteins mimics prenyl pyrophosphates. The 20.1 mAb stimulated Vγ2Vδ2 T cell clones regardless of their functional phenotype or developmental origin and selectively expanded blood Vγ2Vδ2 T cells. The γδ TCR mediates 20.1 mAb stimulation because IL-2 is released by ß(-) Jurkat cells transfected with Vγ2Vδ2 TCRs. 20.1 stimulation was not due to isopentenyl pyrophosphate (IPP) accumulation because 20.1 treatment of APC did not increase IPP levels. In addition, stimulation was not inhibited by statin treatment, which blocks IPP production. Importantly, small interfering RNA knockdown of BTN3A1 abolished stimulation by IPP that could be restored by re-expression of BTN3A1 but not by BTN3A2 or BTN3A3. Rhesus monkey and baboon APC presented HMBPP and 20.1 to human Vγ2Vδ2 T cells despite amino acid differences in BTN3A1 that localize to its outer surface. This suggests that the conserved inner and/or top surfaces of BTN3A1 interact with its counterreceptor. Although no binding site exists on the BTN3A1 extracellular domains, a model of the intracellular B30.2 domain predicts a basic pocket on its binding surface. However, BTN3A1 did not preferentially bind a photoaffinity prenyl pyrophosphate. Thus, BTN3A1 is required for stimulation by prenyl pyrophosphates but does not bind the intermediates with high affinity.
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Antígenos CD/inmunología , Antígenos CD/metabolismo , Hemiterpenos/inmunología , Compuestos Organofosforados/inmunología , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD/genética , Sitios de Unión de Anticuerpos , Butirofilinas , Línea Celular , Células HeLa , Humanos , Interleucina-2/metabolismo , Activación de Linfocitos , Macaca mulatta , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/inmunología , Datos de Secuencia Molecular , Papio , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Alineación de Secuencia , Linfocitos T/inmunología , TerpenosRESUMEN
Despite the enormous therapeutic potential of siRNAs, their delivery is still problematic due to unfavorable biodistribution profiles and poor intracellular bioavailability. Calcium phosphate co-precipitate has been used for nearly 40 years for in vitro transfection due to its non-toxic nature and simplicity of preparation. However, rapid particle growth has largely prevented the translation of this method for in vivo purposes. It has recently been shown that bisphosphonate derivatives can physically stabilize calcium phosphate nanoparticles while still allowing for efficient cell transfection with plasmid DNA. Herein, two novel PEGylated chelating agents (PEG-alendronate and PEG-inositolpentakisphosphate) with enhanced stabilizing properties are introduced, and it is demonstrated that the bisphosphonate-stabilized nanoparticles can efficiently deliver siRNA in vitro. The nanoparticles are mainly taken up by clathrin-dependent endocytosis, and acidification of the endosomal compartment is required to release the entrapped siRNA into the cytosol. Furthermore, particle uptake enhances the inhibition of the mevalonate pathway by the bisphosphonate in macrophages.
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Fosfatos de Calcio/química , Nanocápsulas/administración & dosificación , Nanocápsulas/química , Neoplasias de la Próstata/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Transfección/métodos , Animales , Línea Celular Tumoral , Quelantes/química , Excipientes/química , Humanos , Masculino , Nanocápsulas/ultraestructura , Tamaño de la PartículaRESUMEN
PURPOSE: Nitrogen-containing bisphosphonates (N-BP) such as zoledronate and risedronate exhibit antitumor effects. They block the activity of farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway, leading to intracellular accumulation of mevalonate metabolites (IPP/ApppI), which are recognized as tumor phosphoantigens by Vγ9Vδ2 T cells. However, mechanisms responsible for Vγ9Vδ2 T-cell recognition of N-BP-treated tumors producing IPP/ApppI remain unclear. EXPERIMENTAL DESIGN: The effects of N-BPs on Vγ9Vδ2 T-cell expansion and anticancer activity were evaluated in vitro and in animal models of human breast cancers. The modalities of recognition of breast tumors by Vγ9Vδ2 T cells in N-BP-treated animals were also examined. RESULTS: We found a strong correlation between Vγ9Vδ2 T-cell anticancer activity and intracellular accumulation of IPP/ApppI in risedronate-treated breast cancer cells in vitro. In addition, following risedronate treatment of immunodeficient mice bearing human breast tumors, human Vγ9Vδ2 T cells infiltrated and inhibited growth of tumors that produced high IPP/ApppI levels but not those expressing low IPP/ApppI levels. The combination of doxorubicin with a N-BP improved, however, Vγ9Vδ2 T-cell cytotoxicity against breast tumors expressing low IPP/ApppI levels. Moreover, Vγ9Vδ2 T-cell cytotoxicity in mice treated with risedronate or zoledronate did not only depend on IPP/ApppI accumulation in tumors but also on expression of tumor cell surface receptor intercellular adhesion molecule-1 (ICAM-1), which triggered the recognition of N-BP-treated breast cancer cells by Vγ9Vδ2 T cells in vivo. CONCLUSION: These findings suggest that N-BPs can have an adjuvant role in cancer therapy by activating Vγ9Vδ2 T-cell cytotoxicity in patients with breast cancer that produces high IPP/ApppI levels after N-BP treatment.
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Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Ácido Etidrónico/análogos & derivados , Molécula 1 de Adhesión Intercelular/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T Citotóxicos/metabolismo , Animales , Antígenos de Neoplasias/inmunología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citotoxicidad Inmunológica/efectos de los fármacos , Ácido Etidrónico/farmacología , Femenino , Geraniltranstransferasa/metabolismo , Hemiterpenos/inmunología , Hemiterpenos/metabolismo , Humanos , Factores Inmunológicos/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Compuestos Organofosforados/inmunología , Compuestos Organofosforados/metabolismo , Ácido Risedrónico , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In this study, avidin-biotin technology was combined with a multifunctional drug carrier modality i.e. liposomes to achieve an active and versatile targeting approach. The anti-cancer drug doxorubicin (DOX) was modified with direct biotinylation (B-DOX) (Allart et al., 2003), or encapsulated in biotinylated sterically stabilized pH-sensitive liposomes (BL-DOX), and targeted to the lentiviral vector transduced cells expressing an avidin fusion protein on the cell membrane (Lehtolainen et al., 2003; Lesch et al., 2009). The direct biotinylation of doxorubicin improved cell internalization in rat glioma (BT4C) cells expressing avidin fusion protein receptor but cell toxicity was reduced by 78-fold due to impaired nuclear localization. In contrast, liposomal formulations restored the biological activity of the DOX in several cell lines. However, mainly due to uptake via non-specific pathways the active targeting of BL-DOX was negligible in both in vitro and in vivo studies. Active targeting with multifunctional drug carrier systems is challenging and further studies will be needed to optimize the properties of targeted drug carrier and receptor expression systems.
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Antibióticos Antineoplásicos/administración & dosificación , Avidina/administración & dosificación , Biotina/administración & dosificación , Doxorrubicina/administración & dosificación , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacocinética , Avidina/genética , Biotina/genética , Biotinilación , Línea Celular Tumoral , Doxorrubicina/química , Doxorrubicina/farmacocinética , Humanos , Cinética , Liposomas , Ratones , Ratones Desnudos , Ratas , Proteínas Recombinantes de Fusión/administración & dosificación , Distribución TisularRESUMEN
Curcumin, a natural phytoconstituent, is known to be therapeutically effective in the treatment of various cancers such as, breast cancer, lung cancer, pancreatic cancer, brain cancer, etc. However, low bioavailability and photodegradation of curcumin hampers its overall therapeutic efficacy. Anionic polymerization method was employed for the preparation of apolipoprotein-E3 mediated curcumin loaded poly(butyl)cyanoacrylate nanoparticles (ApoE3-C-PBCA) and characterized for size, zeta potential, entrapment efficiency, photostability, morphology, and in vitro release study. ApoE3-C-PBCA were found to be effective against SH-SY5Y neuroblastoma cells compared to curcumin solution (CSSS) and curcumin loaded PBCA nanoparticles (C-PBCA) from in vitro cell culture investigations. Flow cytometry techniques employed for the detection of anticancer activity revealed enhanced activity of curcumin against SH-SY5Y neuroblastoma cells with ApoE3-C-PBCA compared to CSSS and C-PBCA, and apoptosis being the underlying mechanism. Present study revealed that ApoE3-C-PBCA has tremendous potential to develop into an effective therapeutic treatment modality against brain cancer.
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Antineoplásicos/administración & dosificación , Apolipoproteína E3/administración & dosificación , Curcumina/administración & dosificación , Portadores de Fármacos/administración & dosificación , Enbucrilato/administración & dosificación , Nanopartículas/administración & dosificación , Antineoplásicos/química , Apolipoproteína E3/química , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Curcumina/química , Portadores de Fármacos/química , Enbucrilato/química , Humanos , Ligandos , Nanopartículas/química , Neuroblastoma , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Human peripheral Vγ9Vδ2 T cells are activated by phosphorylated metabolites (phosphoagonists [PAg]) of the mammalian mevalonate or the microbial desoxyxylulose-phosphate pathways accumulated by infected or metabolically distressed cells. The underlying mechanisms are unknown. We show that treatment of nonsusceptible target cells with antibody 20.1 against CD277, a member of the extended B7 superfamily related to butyrophilin, mimics PAg-induced Vγ9Vδ2 T-cell activation and that the Vγ9Vδ2 T-cell receptor is implicated in this effect. Vγ9Vδ2 T-cell activation can be abrogated by exposing susceptible cells (tumor and mycobacteria-infected cells, or aminobisphosphonate-treated cells with up-regulated PAg levels) to antibody 103.2 against CD277. CD277 knockdown and domain-shuffling approaches confirm the key implication of the CD277 isoform BTN3A1 in PAg sensing by Vγ9Vδ2 T cells. Fluorescence recovery after photobleaching (FRAP) experiments support a causal link between intracellular PAg accumulation, decreased BTN3A1 membrane mobility, and ensuing Vγ9Vδ2 T-cell activation. This study demonstrates a novel role played by B7-like molecules in human γδ T-cell antigenic activation and paves the way for new strategies to improve the efficiency of immunotherapies using Vγ9Vδ2 T cells.
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Antígenos CD/metabolismo , Antígenos/metabolismo , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/metabolismo , Anticuerpos Bloqueadores , Anticuerpos Inmovilizados , Anticuerpos Monoclonales , Antígenos CD/química , Antígenos CD/genética , Butirofilinas , Células Cultivadas , Células Clonales , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Factores Inmunológicos/farmacología , Activación de Linfocitos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño , Receptores de Antígenos de Linfocitos T/agonistas , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Broad spectrum therapeutic potential of curcumin is usually hampered by its photodegradation and low bioavailability. Present investigation was designed with an objective to develop transferrin-mediated solid lipid nanoparticles (Tf-C-SLN) resistant to the photostability and capable of enhancing the bioavailability by targeted drug delivery to elicit anticancer activity against SH-SY5Y neuroblastoma cells in vitro. Hot homogenization method was used for the formulation of Tf-C-SLN and evaluated physicochemically using parameters such as, size, zeta potential, entrapment efficiency and photostability, transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), differential scanning colorimetry (DSC), and in vitro release study. In vitro cytotoxicity and apoptosis investigations were performed using microplate analysis and flow cytometry techniques. The physicochemical characterization confirmed the suitability of formulation method and various parameters therein. TEM investigation revealed the spherical morphology while NMR and DSC study confirmed the entrapment of curcumin inside the nanoparticles. The cytotoxicity, reactive oxygen species, and cell uptake were found to be increased considerably with Tf-C-SLN compared with curcumin-solubilized surfactant solution, and curcumin-loaded SLN (C-SLN) suggesting the targeting effect. AnnexinV-FITC/PI double staining, DNA analysis, caspase detection, and reduced mitochondrial potential confirmed the induction of apoptosis with nanoparticle treatment. Enhanced anticancer activity with Tf-C-SLN compared with curcumin-solubilized surfactant solution and C-SLN was observed from flow cytometry investigations with apoptosis being the major underlying mechanism. The in vitro observations of our investigation are very compelling and concrete to advocate the potential of Tf-C-SLN in enhancing the anticancer effect of curcumin against neuroblastoma in vivo and possible clinical applications.
RESUMEN
PURPOSE: For nanocarrier-based targeted delivery systems, preventing phagocytosis for prolong circulation half life is a crucial task. PEGylated poly(n-butylcyano acrylate) (PBCA) NP has proven a promising approach for drug delivery, but an easy and reliable method of PEGylation of PBCA has faced a major bottleneck. METHODS: PEGylated PBCA NPs containing docetaxel (DTX) by modified anionic polymerization reaction in aqueous acidic media containing amine functional PEG were made as an single step PEGylation method. In vitro colloidal stability studies using salt aggregation method and antiopsonization property of prepared NPs using mouse macrophage cell line RAW264 were performed. In vitro performance of anticancer activity of prepared formulations was checked on MCF7 cell line. NPs were radiolabeled with 99mTc and intravenously administered to study blood clearance and biodistribution in mice model. RESULTS: These formulations very effectively prevented phagocytosis and found excellent carrier for drug delivery purpose. In vivo studies display long circulation half life of PBCA-PEG20 NP in comparison to other formulations tested. CONCLUSIONS: The PEGylated PBCA formulation can work as a novel tool for drug delivery which can prevent RES uptake and prolong circulation half life.
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Portadores de Fármacos/farmacocinética , Enbucrilato/farmacocinética , Nanopartículas , Polietilenglicoles/farmacocinética , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Docetaxel , Portadores de Fármacos/química , Enbucrilato/química , Femenino , Semivida , Humanos , Macrófagos/metabolismo , Ratones , Fagocitosis/efectos de los fármacos , Polietilenglicoles/química , Taxoides/administración & dosificación , Tecnecio/química , Distribución TisularRESUMEN
In spite of good research in drug delivery, bone targeting remains largely unexplored. Even some of the bone diseases are seldom cured just because of poor distribution of drug at the bone site. Zoledronate (ZOL) having strong affinity towards bone and its utility in bone metastasis management makes it perfect ligand for bone targeting. Recent studies revealed that ZOL in combination with docetaxel showed significant synergism in the management of bone metastasis. From the results, it is clear that ZOL-conjugated PLGA nanoparticles (NPs) showed more cellular uptake than pegylated PLGA NPs with change in cellular uptake route. In vitro studies on MCF-7 and BO2 cell line revealed that ZOL anchored PLGA-PEG NPs showed enhanced cell cytotoxicity, increase in cell cycle arrest and more apoptotic activity. PLGA-PEG-ZOL NPs found to block mevalonate pathway and increase accumulation of apoptotic metabolites such as ApppI. In vivo animal studies using technetium-99m radiolabeling showed prolong blood circulation half-life, reduced liver uptake and significantly higher retention of ZOL tagged NPs at the bone site with enhanced tumor retention. Here, we can conclude that the targeting ability of ZOL enhanced by strong affinity to bone, enhanced endocytosis of ZOL anchored PLGA-PEG NPs.
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Antineoplásicos/administración & dosificación , Conservadores de la Densidad Ósea/administración & dosificación , Difosfonatos/administración & dosificación , Portadores de Fármacos/administración & dosificación , Imidazoles/administración & dosificación , Ácido Láctico/administración & dosificación , Ácido Poliglicólico/administración & dosificación , Taxoides/administración & dosificación , Animales , Antineoplásicos/farmacocinética , Conservadores de la Densidad Ósea/farmacocinética , Neoplasias Óseas/tratamiento farmacológico , Línea Celular , Línea Celular Tumoral , Difosfonatos/farmacocinética , Docetaxel , Portadores de Fármacos/farmacocinética , Humanos , Imidazoles/farmacocinética , Ácido Láctico/farmacocinética , Ratones , Nanopartículas/administración & dosificación , Metástasis de la Neoplasia , Polietilenglicoles/administración & dosificación , Polietilenglicoles/farmacocinética , Ácido Poliglicólico/farmacocinética , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Taxoides/farmacocinética , Ácido ZoledrónicoRESUMEN
Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the 2-C-methyl-d-erythritol-4-phosphate pathway used by microbes, and isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by humans. Aminobisphosphonates and alkylamines indirectly stimulate Vγ2Vδ2 cells by inhibiting farnesyl diphosphate synthase (FDPS) in the mevalonate pathway, thereby increasing IPP/triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester that directly stimulate. In this study, we further characterize stimulation by these compounds and define pathways used by new classes of compounds. Consistent with FDPS inhibition, stimulation of Vγ2Vδ2 cells by aminobisphosphonates and alkylamines was much more sensitive to statin inhibition than stimulation by prenyl pyrophosphates; however, the continuous presence of aminobisphosphonates was toxic for T cells and blocked their proliferation. Aminobisphosphonate stimulation was rapid and prolonged, independent of known Ag-presenting molecules, and resistant to fixation. New classes of stimulatory compounds-mevalonate, the alcohol of HMBPP, and alkenyl phosphonates-likely stimulate differently. Mevalonate, a rate-limiting metabolite, appears to enter cells to increase IPP levels, whereas the alcohol of HMBPP and alkenyl phosphonates are directly recognized. The critical chemical feature of bisphosphonates is the amino moiety, because its loss switched aminobisphosphonates to direct Ags. Transfection of APCs with small interfering RNA downregulating FDPS rendered them stimulatory for Vγ2Vδ2 cells and increased cellular IPP. Small interfering RNAs for isopentenyl diphosphate isomerase functioned similarly. Our results show that a variety of manipulations affecting isoprenoid metabolism lead to stimulation of Vγ2Vδ2 T cells and that pulsing aminobisphosphonates would be more effective for the ex vivo expansion of Vγ2Vδ2 T cells for adoptive cancer immunotherapy.
Asunto(s)
Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/biosíntesis , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Terpenos/metabolismo , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Proliferación Celular/efectos de los fármacos , Células Clonales , Difosfonatos/metabolismo , Difosfonatos/toxicidad , Relación Dosis-Respuesta Inmunológica , Inhibidores de Crecimiento/metabolismo , Inhibidores de Crecimiento/farmacología , Humanos , Activación de Linfocitos/efectos de los fármacos , Ácido Mevalónico/metabolismo , Ácido Mevalónico/toxicidad , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/efectos de los fármacos , Terpenos/toxicidadRESUMEN
The nitrogen-containing bisphosphonate zoledronic acid (ZOL), a potent inhibitor of farnesyl pyrophosphate synthase, blocks the mevalonate pathway, leading to intracellular accumulation of isopentenyl pyrophosphate/triphosphoric acid I-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (IPP/ApppI) mevalonate metabolites. IPP/ApppI accumulation in ZOL-treated cancer cells may be recognized by Vγ9Vδ2 T cells as tumor phosphoantigens in vitro. However, the significance of these findings in vivo remains largely unknown. In this study, we investigated the correlation between the anticancer activities of Vγ9Vδ2 T cells and the intracellular IPP/ApppI levels in ZOL-treated breast cancer cells in vitro and in vivo. We found marked differences in IPP/ApppI production among different human breast cancer cell lines post-ZOL treatment. Coculture with purified human Vγ9Vδ2 T cells led to IPP/ApppI-dependent near-complete killing of ZOL-treated breast cancer cells. In ZOL-treated mice bearing subcutaneous breast cancer xenografts, Vγ9Vδ2 T cells infiltrated and inhibited growth of tumors that produced high IPP/ApppI levels, but not those expressing low IPP/ApppI levels. Moreover, IPP/ApppI not only accumulated in cancer cells but it was also secreted, promoting Vγ9Vδ2 T-cell chemotaxis to the tumor. Without Vγ9Vδ2 T-cell expansion, ZOL did not inhibit tumor growth. These findings suggest that cancers-producing high IPP/ApppI levels after ZOL treatment are most likely to benefit from Vγ9Vδ2 T-cell-mediated immunotherapy.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Difosfonatos/farmacología , Imidazoles/farmacología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Antígenos de Neoplasias/inmunología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Citotoxicidad Inmunológica , Femenino , Hemiterpenos/metabolismo , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Compuestos Organofosforados/metabolismo , Fosforilación , Linfocitos T/efectos de los fármacos , Ácido ZoledrónicoRESUMEN
A class of drugs successfully used for treatment of metabolic bone diseases is the nitrogen-containing bisphosphonates (N-BPs), which act by inhibiting the vital enzyme, farnesyl pyrophosphate synthase (FPPS), of the mevalonate pathway. Inhibition of FPPS by N-BPs results in the intracellular accumulation of isopentenyl pyrophosphate (IPP) and consequently induces the biosynthesis of a cytotoxic ATP analog (ApppI). Previous cell-free data has reported that N-BPs inhibit FPPS by time-dependent manner as a result of the conformational change. This associated conformational change can be measured as an isomerization constant (K(isom)) and reflects the binding differences of the N-BPs to FPPS. In the present study, we tested the biological relevance of the calculated K(isom) values of zoledronic acid, risedronate and five experimental N-BP analogs in the cell culture model. We used IPP/ApppI formation as a surrogate marker for blocking of FPPS in the mevalonate pathway. As a result, a correlation between the time-dependent inhibition of FPPS and IPP/ApppI formation by N-BPs was observed. This outcome indicates that the time-dependent inhibition of FPPS enzyme is a biologically significant mechanism and further supports the use of the K(isom) calculations for evaluation of the overall potency of the novel FPPS inhibitors. Additionally, data illustrates that IPP/ApppI analysis is a useful method to monitor the intracellular action of drugs and drug candidates based on FPPS inhibition.
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Dimetilaliltranstransferasa/antagonistas & inhibidores , Difosfonatos/farmacología , Ácido Mevalónico/metabolismo , Células Cultivadas , Dimetilaliltranstransferasa/química , Difosfonatos/química , Humanos , Nitrógeno/química , Factores de TiempoRESUMEN
This review describes the key discoveries over the last 15 years that have led to a clearer understanding of the molecular mechanisms by which bisphosphonate drugs inhibit bone resorption. Once released from bone mineral surfaces during bone resorption, these agents accumulate intracellularly in osteoclasts. Simple bisphosphonates such as clodronate are incorporated into non-hydrolysable analogues of adenosine triphosphate, which induce osteoclast apoptosis. The considerably more potent nitrogen-containing bisphosphonates are not metabolised but potently inhibit farnesyl pyrophosphate (FPP) synthase, a key enzyme of the mevalonate pathway. This prevents the synthesis of isoprenoid lipids necessary for the post-translational prenylation of small GTPases, thereby disrupting the subcellular localisation and normal function of these essential signalling proteins. Inhibition of FPP synthase also results in the accumulation of the upstream metabolite isopentenyl diphosphate, which is incorporated into the toxic nucleotide metabolite ApppI. Together, these properties explain the ability of bisphosphonate drugs to inhibit bone resorption by disrupting osteoclast function and survival. These discoveries are also giving insights into some of the adverse effects of bisphosphonates, such as the acute phase reaction that is triggered by inhibition of FPP synthase in peripheral blood monocytes.