RESUMEN
Biodegradable periodic mesoporous organosilica (BPMO) has recently emerged as a promising type of mesoporous silica-based nanoparticle for biomedical applications. Like mesoporous silica nanoparticles (MSN), BPMO possesses a large surface area where various compounds can be attached. In this work, we attached boronophenylalanine (10BPA) to the surface and explored the potential of this nanomaterial for delivering boron-10 for use in boron neutron capture therapy (BNCT). This cancer therapy is based on the principle that the exposure of boron-10 to thermal neutron results in the release of a-particles that kill cancer cells. To attach 10BPA, the surface of BPMO was modified with diol groups which facilitated the efficient binding of 10BPA, yielding 10BPA-loaded BPMO (10BPA-BPMO). Surface modification with phosphonate was also carried out to increase the dispersibility of the nanoparticles. To investigate this nanomaterial's potential for BNCT, we first used human cancer cells and found that 10BPA-BPMO nanoparticles were efficiently taken up into the cancer cells and were localized in perinuclear regions. We then used a chicken egg tumor model, a versatile and convenient tumor model used to characterize nanomaterials. After observing significant tumor accumulation, 10BPA-BPMO injected chicken eggs were evaluated by irradiating with neutron beams. Dramatic inhibition of the tumor growth was observed. These results suggest the potential of 10BPA-BPMO as a novel boron agent for BNCT.
Asunto(s)
Antineoplásicos/administración & dosificación , Antineoplásicos/química , Compuestos de Boro/química , Nanopartículas del Metal/administración & dosificación , Neoplasias/tratamiento farmacológico , Compuestos de Organosilicio/química , Fenilalanina/química , Apoptosis , Proliferación Celular , Humanos , Nanopartículas del Metal/química , Neoplasias/patología , Células Tumorales CultivadasRESUMEN
BACKGROUND: RHEB is a unique member of the RAS superfamily of small GTPases expressed in all tissues and conserved from yeast to humans. Early studies on RHEB indicated a possible RHEB-RAF interaction, but this has not been fully explored. Recent work on cancer genome databases has revealed a reoccurring mutation in RHEB at the Tyr35 position, and a recent study points to the oncogenic potential of this mutant that involves activation of RAF/MEK/ERK signaling. These developments prompted us to reassess the significance of RHEB effect on RAF, and to compare mutant and wild type RHEB. METHODS: To study RHEB-RAF interaction, and the effect of the Y35N mutation on this interaction, we used transfection, immunoprecipitation, and Western blotting techniques. We generated cell lines stably expressing RHEB WT, RHEB Y35N, and KRAS G12V, and monitored cellular transforming properties through cell proliferation, anchorage independent growth, cell cycle analysis, and foci formation assays. RESULTS: We observe a strong interaction between RHEB and BRAF, but not with CRAF. This interaction is dependent on an intact RHEB effector domain and RHEB-GTP loading status. RHEB overexpression decreases RAF activation of the RAF/MEK/ERK pathway and RHEB knockdown results in an increase in RAF/MEK/ERK activation. RHEB Y35N mutation has decreased interaction with BRAF, and RHEB Y35N cells exhibit greater BRAF/CRAF heterodimerization resulting in increased RAF/MEK/ERK signaling. This leads to cancer transformation of RHEB Y35N stably expressing cell lines, similar to KRAS G12 V expressing cell lines. CONCLUSIONS: RHEB interaction with BRAF is crucial for inhibiting RAF/MEK/ERK signaling. The RHEB Y35N mutant sustains RAF/MEK/ERK signaling due to a decreased interaction with BRAF, leading to increased BRAF/CRAF heterodimerization. RHEB Y35N expressing cells undergo cancer transformation due to decreased interaction between RHEB and BRAF resulting in overactive RAF/MEK/ERK signaling. Taken together with the previously established function of RHEB to activate mTORC1 signaling, it appears that RHEB performs a dual function; one is to suppress the RAF/MEK/ERK signaling and the other is to activate mTORC1 signaling.
Asunto(s)
Carcinogénesis/genética , Neoplasias/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteína Homóloga de Ras Enriquecida en el Cerebro/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Sistema de Señalización de MAP Quinasas/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Mutación , Neoplasias/patología , Fosforilación , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
TWIST protein is critical to development and is activated in many cancers. TWIST regulates epithelial-mesenchymal transition, and is linked to angiogenesis, metastasis, cancer stem cell phenotype, and drug resistance. The majority of epithelial ovarian cancer (EOC) patients with metastatic disease respond well to first-line chemotherapy but most relapse with disease that is both metastatic and drug resistant, leading to a five-year survival rate under 20%. We are investigating the role of TWIST in mediating these relapses. We demonstrate TWIST-siRNA (siTWIST) and a novel nanoparticle delivery platform to reverse chemoresistance in an EOC model. Hyaluronic-acid conjugated mesoporous silica nanoparticles (MSN-HAs) carried siTWIST into target cells and led to sustained TWIST knockdown in vitro. Mice treated with siTWIST-MSN-HA and cisplatin exhibited specific tumor targeting and reduction of tumor burden. This platform has potential application for overcoming clinical challenges of tumor cell targeting, metastasis and chemoresistance in ovarian and other TWIST overexpressing cancers.
Asunto(s)
Cisplatino/uso terapéutico , Ácido Hialurónico/química , Nanopartículas/química , Neoplasias Ováricas/tratamiento farmacológico , ARN Interferente Pequeño/química , Animales , Western Blotting , Línea Celular Tumoral , Femenino , Humanos , Ratones , Microscopía Confocal , Microscopía Fluorescente , Neoplasias Ováricas/metabolismo , ARN Interferente Pequeño/administración & dosificación , Carga Tumoral/efectos de los fármacos , Factores de Transcripción Twist/genética , Factores de Transcripción Twist/metabolismoRESUMEN
Protein prenylation such as farnesylation and geranylgeranylation is associated with various diseases. Thus, many inhibitors of prenyltransferase have been developed. We report novel inhibitors of farnesyltransferase with a zinc-site recognition moiety and a farnesyl/dodecyl group. Molecular docking analysis showed that both parts of the inhibitor fit well into the catalytic domain of farnesyltransferase. The synthesized inhibitors showed activity against farnesyltransferase in vitro and inhibited proliferation of the pancreatic cell line AsPC-1. Among the compounds with farnesyl and dodecyl groups, the inhibitor with a farnesyl group was found to have stronger and more selective activity.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Compuestos Organometálicos/farmacología , Zinc/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Farnesiltransferasa/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Relación Estructura-Actividad , Zinc/químicaRESUMEN
Epithelial ovarian cancer (EOC) is the most deadly gynecologic malignancy on account of its late stage at diagnosis and frequency of drug resistant recurrences. Novel therapies to overcome these barriers are urgently needed. TWIST is a developmental transcription factor reactivated in cancers and linked to angiogenesis, metastasis, cancer stem cell phenotype, and drug resistance, making it a promising therapeutic target. In this work, we demonstrate the efficacy of TWIST siRNA (siTWIST) and two nanoparticle delivery platforms to reverse chemoresistance in EOC models. Polyamidoamine dendrimers and mesoporous silica nanoparticles (MSNs) carried siTWIST into target cells and led to sustained TWIST knockdown in vitro. Mice treated with cisplatin plus MSN-siTWIST exhibited lower tumor burden than mice treated with cisplatin alone, with most of the effect coming from reduction in disseminated tumors. This platform has potential application for overcoming the clinical challenges of metastasis and chemoresistance in EOC and other TWIST overexpressing cancers.
Asunto(s)
Nanopartículas/química , Neoplasias Glandulares y Epiteliales/terapia , Neoplasias Ováricas/terapia , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/uso terapéutico , Tratamiento con ARN de Interferencia/métodos , Dióxido de Silicio/química , Proteína 1 Relacionada con Twist/genética , Animales , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Dendrímeros/química , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Nanopartículas/ultraestructura , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Ovario/metabolismo , Ovario/patología , Porosidad , ARN Interferente Pequeño/genéticaRESUMEN
Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis.
Asunto(s)
Aspartato Carbamoiltransferasa/metabolismo , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/metabolismo , Dihidroorotasa/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Neuropéptidos/metabolismo , Nucleósidos de Pirimidina/biosíntesis , Proteínas ras/metabolismo , Animales , Proliferación Celular/genética , Humanos , Lisosomas/metabolismo , Lisosomas/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Proteínas de Unión al GTP Monoméricas/genética , Complejos Multiproteicos/metabolismo , Neuropéptidos/genética , Unión Proteica , Proteína Homóloga de Ras Enriquecida en el Cerebro , Serina-Treonina Quinasas TOR/metabolismo , Proteínas ras/genéticaRESUMEN
Despite the worldwide interest generated by periodic mesoporous organosilica (PMO) bulk materials, the design of PMO nanomaterials with controlled morphology remains largely unexplored and their properties unknown. In this work, we describe the first study of PMO nanoparticles (NPs) based on meta-phenylene bridges, and we conducted a comparative structure-property relationship investigation with para-phenylene-bridged PMO NPs. Our findings indicate that the change of the isomer drastically affects the structure, morphology, size, porosity and thermal stability of PMO materials. We observed a much higher porosity and thermal stability of the para-based PMO which was likely due to a higher molecular periodicity. Additionally, the para isomer could generate multipodal NPs at very low stirring speed and upon this discovery we designed a phenylene-ethylene bridged PMO with a controlled Janus morphology. Unprecedentedly high payloads could be obtained from 40 to 110â wt % regardless of the organic bridge of PMOs. Finally, we demonstrate for the first time the co-delivery of two cargos by PMO NPs. Importantly, the cargo stability in PMOs did not require the capping of the pores, unlike pure silica, and the delivery could be autonomously triggered in cancer cells by acidic pH with nearly 70 % cell killing.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/química , Nanoestructuras/química , Compuestos de Organosilicio/química , Nanopartículas/uso terapéutico , Porosidad , Propiedades de SuperficieRESUMEN
We describe biodegradable mesoporous hybrid nanoparticles (NPs) in the presence of proteins and their applications for drug delivery. We synthesized oxamide phenylene-based mesoporous organosilica nanoparticles (MON) in the absence of a silica source which had remarkably high organic content and high surface areas. Oxamide functions provided biodegradability in the presence of trypsin model proteins. MON displayed exceptionally high payloads of hydrophilic and hydrophobic drugs (up to 84â wt %), and a unique zero premature leakage without the pore capping, unlike mesoporous silica. MON were biocompatible and internalized into cancer cells for drug delivery.
Asunto(s)
Nanopartículas/química , Compuestos de Organosilicio/química , Ácido Oxámico/análogos & derivados , Dióxido de Silicio/química , Sistemas de Liberación de Medicamentos , Interacciones Hidrofóbicas e Hidrofílicas , Ácido Oxámico/químicaRESUMEN
mTORC1 plays critical roles in the regulation of protein synthesis, growth, and proliferation in response to nutrients, growth factors, and energy conditions. One of the substrates of mTORC1 is 4E-BP1, whose phosphorylation by mTORC1 reverses its inhibitory action on eIF4E, resulting in the promotion of protein synthesis. Raptor in mTOR complex 1 is believed to recruit 4E-BP1, facilitating phosphorylation of 4E-BP1 by the kinase mTOR. We applied chemical cross-linking coupled with mass spectrometry analysis to gain insight into interactions between mTORC1 and 4E-BP1. Using the cross-linking reagent bis[sulfosuccinimidyl] suberate, we showed that Raptor can be cross-linked with 4E-BP1. Mass spectrometric analysis of cross-linked Raptor-4E-BP1 led to the identification of several cross-linked peptide pairs. Compilation of these peptides revealed that the most N-terminal Raptor N-terminal conserved domain (in particular residues from 89 to 180) of Raptor is the major site of interaction with 4E-BP1. On 4E-BP1, we found that cross-links with Raptor were clustered in the central region (amino acid residues 56-72) we call RCR (Raptor cross-linking region). Intramolecular cross-links of Raptor suggest the presence of two structured regions of Raptor: one in the N-terminal region and the other in the C-terminal region. In support of the idea that the Raptor N-terminal conserved domain and the 4E-BP1 central region are closely located, we found that peptides that encompass the RCR of 4E-BP1 inhibit cross-linking and interaction of 4E-BP1 with Raptor. Furthermore, mutations of residues in the RCR decrease the ability of 4E-BP1 to serve as a substrate for mTORC1 in vitro and in vivo.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Espectrometría de Masas/métodos , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Secuencia Conservada , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lisina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Mutación/genética , Péptidos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Ratas , Proteína Reguladora Asociada a mTOR , Especificidad por Sustrato/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
BACKGROUND: Glioblastoma multiforme (GBM) is one of the most highly metastatic cancers. GBM has been associated with a high level of the mechanistic target of rapamycin complex 2 (mTORC2) activity. We aimed to observe roles of mTORC2 in GBM cells especially on actin cytoskeleton reorganization, cell migration and invasion, and further determine new important players involved in the regulation of these cellular processes. METHODS: To further investigate the significance of mTORC2 in GBM, we treated GBM cells with PP242, an ATP-competitive inhibitor of mTOR, and used RICTOR siRNA to knock down mTORC2 activity. Effects on actin cytoskeleton, focal adhesion, migration, and invasion of GBM cells were examined. To gain insight into molecular basis of the mTORC2 effects on cellular cytoskeletal arrangement and motility/invasion, we affinity purified mTORC2 from GBM cells and identified proteins of interest by mass spectrometry. Characterization of the protein of interest was performed. RESULTS: In addition to the inhibition of mTORC2 activity, we demonstrated significant alteration of actin distribution as revealed by the use of phalloidin staining. Furthermore, vinculin staining was altered which suggests changes in focal adhesion. Inhibition of cell migration and invasion was observed with PP242. Two major proteins that are associated with this mTORC2 multiprotein complex were found. Mass spectrometry identified one of them as Filamin A (FLNA). Association of FLNA with RICTOR but not mTOR was demonstrated. Moreover, in vitro, purified mTORC2 can phosphorylate FLNA likewise its known substrate, AKT. In GBM cells, colocalization of FLNA with RICTOR was observed, and the overall amounts of FLNA protein as well as phosphorylated FLNA are high. Upon treatments of RICTOR siRNA or PP242, phosphorylated FLNA levels at the regulatory residue (Ser2152) decreased. This treatment also disrupted colocalization of Actin filaments and FLNA. CONCLUSIONS: Our results support FLNA as a new downstream effector of mTORC2 controlling GBM cell motility. This new mTORC2-FLNA signaling pathway plays important roles in motility and invasion of glioblastoma cells.
Asunto(s)
Filaminas/metabolismo , Glioblastoma/metabolismo , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Portadoras/metabolismo , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Glioblastoma/patología , Humanos , Indoles/farmacología , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/antagonistas & inhibidores , Fosforilación , Unión Proteica , Purinas/farmacología , Proteína Asociada al mTOR Insensible a la Rapamicina , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
A multifunctional mesoporous drug delivery system that contains fluorescent imaging molecules, targeting proteins, and pH-sensitive nanovalves is developed and tested. Three nanovalve-mesoporous silica nanoparticle (NV-MSN) systems with varied quantities of nanovalves on the surface are synthesized. These systems are characterized and tested to optimize the trade-off between the coverage of nanovalves on the MSNs to effectively trap and deliver cargo, and the remaining underivatized silanol groups that can be used for protein attachments. The NV-MSN system that has satisfactory coverage for high loading and spare silanols is chosen, and transferrin (Tf) is integrated into the system. Abiotic studies are performed to test the operation of the nanovalve in the presence of the protein. In vitro studies are carried out to demonstrate the autonomous activation and function of the nanovalves in the system under biological conditions. Enhanced cellular uptake of the Tf-modified MSNs is seen using fluorescence microscopy and flow cytometry in MiaPaCa-2 cells. The MSNs are then tested using SCID mice, which show that both targeted and untargeted NV-MSN systems are fully functional to effectively deliver cargo. These new multifunctional nanoparticles serve proof of concept of nanovalve functionality in the presence of large proteins and demonstrate another dimension of MSN-based theranostic platforms.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/química , Transferrina/metabolismo , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Preparaciones de Acción Retardada , Doxorrubicina/farmacología , Endocitosis/efectos de los fármacos , Colorantes Fluorescentes/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Espacio Intracelular/metabolismo , Ratones SCID , Microscopía Fluorescente , Nanopartículas/ultraestructura , Porosidad , Dióxido de Silicio/química , Distribución Tisular/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Key effector(s) of mutated KRAS in lung cancer progression and metastasis are unknown. Here we investigated the role of PAK1/Crk axis in transduction of the oncogenic KRAS signal in non-small cell lung cancer (NSCLC). METHODS: We used NSCLC clinical specimens to examine the correlation among KRAS mutations (codon 12, 13 and 61); PAK1/Crk axis activation [p-PAK1(Thr423), p-Crk(Ser41)]; and adhesion molecules expression by immunohistochemistry. For assessing the role of proto-oncogene c-Crk as a KRAS effector, we inhibited KRAS in NSCLC cells by a combination of farnesyltransferase inhibitor (FTI) and geranylgeranyltransferase inhibitor (GGTI) and measured p-Crk-II(Ser41) by western blotting. Finally, we disrupted the signaling network downstream of KRAS by blocking KRAS/PAK1/Crk axis with PAK1 inhibitors (i.e., IPA-3, FRAX597 or FRAX1036) along with partial inhibition of all other KRAS effectors by prenylation inhibitors (FTI + GGTI) and examined the motility, morphology and proliferation of the NSCLC cells. RESULTS: Immunohistochemical analysis demonstrated an inverse correlation between PAK1/Crk phosphorylation and E-cadherin/p120-catenin expression. Furthermore, KRAS mutant tumors expressed higher p-PAK1(Thr423) compared to KRAS wild type. KRAS prenylation inhibition by (FTI + GGTI) completely dephosphorylated proto-oncogene c-Crk on Serine 41 while Crk phosphorylation did not change by individual prenylation inhibitors or diluent. Combination of PAK1 inhibition and partial inhibition of all other KRAS effectors by (FTI + GGTI) dramatically altered morphology, motility and proliferation of H157 and A549 cells. CONCLUSIONS: Our data provide evidence that proto-oncogene c-Crk is operative downstream of KRAS in NSCLC. Previously we demonstrated that Crk receives oncogenic signals from PAK1. These data in conjunction with the work of others that have specified the role of PAK1 in transduction of KRAS signal bring forward the importance of KRAS/PAK1/Crk axis as a prominent pathway in the oncogenesis of KRAS mutant lung cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogénicas c-crk/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Quinasas p21 Activadas/metabolismo , Cadherinas , Carcinogénesis , Carcinoma de Pulmón de Células no Pequeñas/genética , Cateninas , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Neoplasias Pulmonares/genética , Fosforilación , Prenilación , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-crk/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal/fisiología , Quinasas p21 Activadas/genética , Catenina deltaRESUMEN
Growth and progression of solid tumors depend on the integration of multiple pro-growth and survival signals, including the induction of angiogenesis. TWIST1 is a transcription factor whose reactivation in tumors leads to epithelial to mesenchymal transition (EMT), including increased cancer cell stemness, survival, and invasiveness. Additionally, TWIST1 drives angiogenesis via activation of IL-8 and CCL2, independent of VEGF signaling. In this work, results suggest that chemically modified siRNA against TWIST1 reverses EMT both in vitro and in vivo. siRNA delivery with a polyethyleneimine-coated mesoporous silica nanoparticle (MSN) led to reduction of TWIST1 target genes and migratory potential in vitro. In mice bearing xenograft tumors, weekly intravenous injections of the siRNA-nanoparticle complexes resulted in decreased tumor burden together with a loss of CCL2 suggesting a possible anti-angiogenic response. Therapeutic use of TWIST1 siRNA delivered via MSNs has the potential to inhibit tumor growth and progression in many solid tumor types. FROM THE CLINICAL EDITOR: Tumor progression and metastasis eventually lead to patient mortality in the clinical setting. In other studies, it has been found that TWIST1, a transcription factor, if reactivated in tumors, would lead to downstream events including angiogenesis and result in poor prognosis in cancer patients. In this article, the authors were able to show that when siRNA against TWIST1 was delivered via mesoporous silica nanoparticle, there was tumor reduction in an in-vivo model. The results have opened up a new avenue for further research in this field.
Asunto(s)
Nanopartículas/administración & dosificación , Neoplasias/terapia , Neovascularización Patológica/terapia , Proteínas Nucleares/genética , ARN Interferente Pequeño/administración & dosificación , Proteína 1 Relacionada con Twist/genética , Animales , Línea Celular Tumoral , Técnicas de Transferencia de Gen , Humanos , Ratones , Nanopartículas/química , Neoplasias/genética , Neoplasias/patología , Neovascularización Patológica/genética , Proteínas Nucleares/antagonistas & inhibidores , ARN Interferente Pequeño/química , Dióxido de Silicio/administración & dosificación , Dióxido de Silicio/química , Carga Tumoral/genética , Proteína 1 Relacionada con Twist/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Target of rapamycin (TOR), an evolutionarily conserved serine/threonine protein kinase, plays pivotal roles in several important cellular processes in eukaryotes. In the fission yeast Schizosaccharomyces pombe, TOR complex 1 (TORC1), which includes Tor2 as a catalytic subunit, manages the switch between cell proliferation and differentiation by sensing nutrient availability. However, little is known about the direct target of TORC1 that plays key roles in nutrient-dependent TORC1 signaling in fission yeast. Here we report that in fission yeast, three AGC kinase family members, named Psk1, Sck1 and Sck2, which exhibit high homology with human S6K1, are phosphorylated under nutrient-rich conditions and are dephosphorylated by starvation conditions. Among these, Psk1 is necessary for phosphorylation of ribosomal protein S6. Furthermore, Psk1 phosphorylation is regulated by TORC1 in nutrient-dependent and rapamycin-sensitive manners in vivo. Three conserved regulatory motifs (the activation loop, the hydrophobic and the turn motifs) in Psk1 are phosphorylated and these modifications are required for Psk1 activity. In particular, phosphorylation of the hydrophobic motif is catalyzed by TORC1 in vivo and in vitro. Ksg1, a homolog of PDK1, is also important for Psk1 phosphorylation in the activation loop and for its activity. The TORC1 components Pop3, Toc1 and Tco89, are dispensable for Psk1 regulation, but disruption of pop3(+) causes an increase in the sensitivity of TORC1 to rapamycin. Taken together, these results provide convincing evidence that TORC1/Psk1/Rps6 constitutes a nutrient-dependent signaling pathway in fission yeast.
Asunto(s)
Complejos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Schizosaccharomyces/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/genética , Fosforilación/genética , Fosforilación/fisiología , Proteínas Quinasas S6 Ribosómicas/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Mesoporous silica nanoparticles (MSNPs) are functionalized with molecular-recognition sites by anchoring a triazine or uracil fragment on the surface. After loading these MSNPs with dyes (propidium iodide or rhodamine B) or with a drug (camptothecin, CPT) they are capped by the complementary fragments, uracil and adenine, respectively, linked to the bulky cyclodextrin ring. These MSNPs are pH-sensitive and indeed, the dye release was observed at acidic pH by continuously monitored fluorescence spectroscopy studies. On the other hand, no dye leakage occurred at neutral pH, hence meeting the non-premature requirement to minimize side effects. In vitro studies were performed and confocal microscopy images demonstrate the internalization of the MSNPs and also dye release in the cells. To investigate the drug-delivery performance, the cytotoxicity of CPT-loaded nanoparticles was tested and cell death was observed. A remarkably lower amount of loaded CPT in the MSNPs (more than 40 times less) proved to be as efficient as free CPT. These results not only demonstrate the drug release after pore opening under lysosomal pH, but they also show the potential use of these MSNPs to significantly decrease the amount of the administered drug.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/química , Dióxido de Silicio/química , Humanos , Concentración de Iones de Hidrógeno , Células MCF-7 , Microscopía Confocal/métodos , Nanopartículas/administración & dosificación , Dióxido de Silicio/administración & dosificación , Triazinas/química , Uracilo/químicaRESUMEN
The exocytosis of phosphonate modified mesoporous silica nanoparticles (P-MSNs) is demonstrated and lysosomal exocytosis is identified as the mechanism responsible for this event. Regulation of P-MSN exocytosis can be achieved by inhibiting or accelerating lysosomal exocytosis. Slowing down P-MSN exocytosis enhances the drug delivery effect of CPT-loaded P-MSNs by improving cell killing.
Asunto(s)
Portadores de Fármacos/química , Lisosomas/química , Nanopartículas/química , Dióxido de Silicio/química , Línea Celular , Exocitosis/fisiología , HumanosRESUMEN
BACKGROUND: Lung cancer is the leading cause of cancer-related mortality. Therapies against non-small cell lung cancer (NSCLC) are particularly needed, as this type of cancer is relatively insensitive to chemotherapy and radiation therapy. We recently identified GGTI compounds that are designed to block geranylgeranylation and membrane association of signaling proteins including the Rho family G-proteins. One of the GGTIs is P61A6 which inhibits proliferation of human cancer cells, causes cell cycle effects with G1 accumulation and exhibits tumor-suppressing effects with human pancreatic cancer xenografts. In this paper, we investigated effects of P61A6 on non-small cell lung cancer (NSCLC) cells in vitro and in vivo. METHODS: Three non-small cell lung cancer cell lines were used to test the ability of P61A6 to inhibit cell proliferation. Further characterization involved analyses of geranylgeranylation, membrane association and activation of RhoA, and anchorage-dependent and -independent growth, as well as cell cycle effects and examination of cell cycle regulators. We also generated stable cells expressing RhoA-F, which bypasses the geranylgeranylation requirement of wild type RhoA, and examined whether the proliferation inhibition by P61A6 is suppressed in these cells. Tumor xenografts of NSCLC cells growing in nude mice were also used to test P61A6's tumor-suppressing ability. RESULTS: P61A6 was shown to inhibit proliferation of NSCLC lines H358, H23 and H1507. Detailed analysis of P61A6 effects on H358 cells showed that P61A6 inhibited geranylgeranylation, membrane association of RhoA and caused G1 accumulation associated with decreased cyclin D1/2. The effects of P61A6 to inhibit proliferation could mainly be ascribed to RhoA, as expression of the RhoA-F geranylgeranylation bypass mutant rendered the cells resistant to inhibition by P61A6. We also found that P61A6 treatment of H358 tumor xenografts growing in nude mice reduced their growth as well as the membrane association of RhoA in the tumors. CONCLUSION: Thus, P61A6 inhibits proliferation of NSCLC cells and causes G1 accumulation associated with decreased cyclin D1/2. The result with the RhoA-F mutant suggests that the effect of P61A6 to inhibit proliferation is mainly through the inhibition of RhoA. P61A6 also shows efficacy to inhibit growth of xenograft tumor.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Fenilalanina/análogos & derivados , Sulfonamidas/farmacología , Proteína de Unión al GTP rhoA/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclina D1/metabolismo , Ciclina D2/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Neoplasias Pulmonares/enzimología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Fenilalanina/farmacología , Fenilalanina/uso terapéutico , Prenilación/efectos de los fármacos , Sulfonamidas/uso terapéuticoRESUMEN
Microscopy has greatly advanced our understanding of biology. Although significant progress has recently been made in optical microscopy to break the diffraction-limit barrier, reliance of such techniques on fluorescent labeling technologies prohibits quantitative 3D imaging of the entire contents of cells. Cryoelectron microscopy can image pleomorphic structures at a resolution of 3-5 nm, but is only applicable to thin or sectioned specimens. Here, we report quantitative 3D imaging of a whole, unstained cell at a resolution of 50-60 nm by X-ray diffraction microscopy. We identified the 3D morphology and structure of cellular organelles including cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, nucleus, and nucleolus inside a yeast spore cell. Furthermore, we observed a 3D structure protruding from the reconstructed yeast spore, suggesting the spore germination process. Using cryogenic technologies, a 3D resolution of 5-10 nm should be achievable by X-ray diffraction microscopy. This work hence paves a way for quantitative 3D imaging of a wide range of biological specimens at nanometer-scale resolutions that are too thick for electron microscopy.
Asunto(s)
Microscopía por Crioelectrón/métodos , Imagenología Tridimensional/métodos , Schizosaccharomyces/fisiología , Difracción de Rayos X/métodos , Algoritmos , Electrones , Procesamiento de Imagen Asistido por Computador , Modelos Biológicos , Modelos Estadísticos , Óptica y Fotónica , Dispersión de Radiación , Schizosaccharomyces/metabolismo , Esporas Fúngicas/metabolismo , Rayos X , Levaduras/metabolismoRESUMEN
A therapy of cancer cells: Two-photon-triggered camptothecin delivery with nanoimpellers was studied in MCF-7 breast cancer cells. A fluorophore with a high two-photon absorption cross-section was first incorporated in the nanoimpellers. Fluorescence resonance energy transfer (FRET) from the fluorophore to the azobenzene moiety was demonstrated.
Asunto(s)
Compuestos Azo/química , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/química , Humanos , NeoplasiasRESUMEN
Since its publication in 1950, the series "The Enzymes" has been established as an important reference book for researchers and students in the field of enzymology, biochemistry and biophysics and medical research. A number of scientists have served as a series editor for the Enzymes. Topics covered range from characterizations of various enzymes, biochemical processes and medical applications. This chapter provides an overview of the history of The Enzymes.