RESUMEN
Gene therapies represent promising new therapeutic options for a variety of indications. However, despite several approved drugs, its potential remains untapped. For polymeric gene delivery, endosomal escape represents a bottleneck. SO1861, a naturally occurring triterpene saponin with endosomal escape properties isolated from Saponaria officinalis L., has been described as additive agent to enhance transfection efficiency (sapofection). However, the challenge to synchronize the saponin and gene delivery system in vivo imposes limitations. Herein, we address this issue by conjugating SO1861 to a peptide-based gene vector using a pH-sensitive hydrazone linker programmed to release SO1861 at the acidic pH of the endosome. Nanoplexes formulated with SO1861-equipped peptides were investigated for transfection efficiency and tolerability in vitro and in vivo. In all investigated cell lines, SO1861-conjugated nanoplexes have shown superior transfection efficiency and cell viability over supplementation of transfection medium with free SO1861. Targeted SO1861-equipped nanoplexes incorporating a targeting peptide were tested in vitro and in vivo in an aggressively growing neuroblastoma allograft model in mice. Using a suicide gene vector encoding the cytotoxic protein saporin, a slowed tumor growth and improved survival rate were observed for targeted SO1861-equipped nanoplexes compared to vehicle control.
Asunto(s)
Saponinas , Animales , Humanos , Ratones , Saponinas/química , Saponinas/farmacología , Línea Celular Tumoral , Técnicas de Transferencia de Gen , Péptidos/química , Transfección/métodos , Saponaria/química , Saporinas/química , Saporinas/farmacología , Terapia Genética , Supervivencia Celular/efectos de los fármacos , Cationes/químicaRESUMEN
Cellular uptake of vitamin B12 in humans is mediated by the endocytosis of the B12 carrier protein transcobalamin (TC) via its cognate cell surface receptor TCblR, encoded by the CD320 gene. Because CD320 expression is associated with the cell cycle and upregulated in highly proliferating cells including cancer cells, this uptake route is a potential target for cancer therapy. We developed and characterized four camelid nanobodies that bind holo-TC (TC in complex with B12 ) or the interface of the human holo-TC:TCblR complex with nanomolar affinities. We determined X-ray crystal structures of these nanobodies bound to holo-TC:TCblR, which enabled us to map their binding epitopes. When conjugated to the model toxin saporin, three of our nanobodies caused growth inhibition of HEK293T cells and therefore have the potential to inhibit the growth of human cancer cells. We visualized the cellular binding and endocytic uptake of the most potent nanobody (TC-Nb4) using fluorescent light microscopy. The co-crystal structure of holo-TC:TCblR with another nanobody (TC-Nb34) revealed novel features of the interface of TC and the LDLR-A1 domain of TCblR, rationalizing the decrease in the affinity of TC-B12 binding caused by the Δ88 mutation in CD320.
Asunto(s)
Anticuerpos Monoclonales/química , Inmunoconjugados/farmacología , Receptores de Superficie Celular/metabolismo , Saporinas/química , Anticuerpos de Dominio Único/química , Transcobalaminas/metabolismo , Vitamina B 12/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Camélidos del Nuevo Mundo , Ciclo Celular , Proliferación Celular , Células HEK293 , Humanos , Inmunoconjugados/química , Inmunoconjugados/inmunología , Inmunotoxinas/química , Inmunotoxinas/farmacología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Saporinas/inmunología , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/inmunologíaRESUMEN
Dingkun Dan (DKD), a reputable traditional Chinese medicine formula, has been used to treat gynecological diseases and showed significant clinical effects since ancient times. However, the application and development of DKD are seriously hampered by the unclear active substances. Structural characterization of compounds absorbed in vivo and their corresponding metabolites is significant for clarifying the pharmacodynamic material basis. In this study, an integrated strategy using ultra-performance liquid chromatography, coupled with quadrupole time-of-flight mass spectrometry and UNIFI™ software, was used to identify prototypes and metabolites after oral administration of DKD in rats. As a result, a total of 261 compounds, including 140 prototypes and 121 metabolites, were tentatively characterized in rat plasma, urine, and feces. The metabolic pathways of prototypes have been studied to clarify their possible transformation process in vivo. Moreover, an in vitro metabolism study was applied for verifying the metabolites under simulating the metabolic environment in vivo. This first systematic metabolic study of DKD is important for elucidating the metabolites and metabolic pathways and could provide a scientific basis for explaining the integrative mechanism in further pharmacology study.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/metabolismo , Espectrometría de Masas/métodos , Administración Oral , Alcaloides/análisis , Alcaloides/química , Alcaloides/metabolismo , Animales , Medicamentos Herbarios Chinos/administración & dosificación , Flavonoides/análisis , Flavonoides/química , Flavonoides/metabolismo , Redes y Vías Metabólicas , Ratas , Saporinas/análisis , Saporinas/química , Saporinas/metabolismoRESUMEN
For the purpose of efficient targeted therapies, suppressing phagocytosis by a mononuclear phagocyte system (MPS), enhancing the "active" targeted delivery, and meeting clinical production criteria are extremely critical for engineering strategies of novel drug delivery systems. Herein, we used a chemically-induced membrane blebbing and extrusion combined method to induce triple-negative breast cancer (TNBC) cell apoptosis to secrete apoptotic body analogue (ABA) vesicles on a large scale for therapeutic drug delivery. After optimization, the ABAs have a desirable size, good biocompatibility, and long-term colloidal stability. Furthermore, ABAs present anti-phagocytosis ("don't eat me") and specific homologous targeting ("eat me") capacities because of their inheritance of membrane proteins such as CD47 and cellular adhesion molecules from parent cells. After loading with toxic protein saporin and anti-twist siRNA, ABAs can significantly inhibit the growth and lung metastasis of TNBC in an orthotopic metastasis model due to their reduced clearance of immune organs, long circulation time, and enhanced targeted accumulation at the tumor sites. These results suggest the great potential of ABAs for targeted drug delivery therapy, in particular efficient TNBC treatment.
Asunto(s)
Apoptosis , Sistemas de Liberación de Medicamentos , Sistema Mononuclear Fagocítico/fisiología , Neoplasias de la Mama Triple Negativas/terapia , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Vesículas Extracelulares , Humanos , Ratones , Ratones Endogámicos ICR , Ratones Desnudos , Nanoestructuras , Fagocitosis , Células RAW 264.7 , Distribución Aleatoria , Saporinas/química , Saporinas/farmacología , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Intracellular delivery of therapeutic proteins remains a challenge for the success of protein-mediated disease treatment. We herein develop a robust nanoplatform made with a TME-pH responsive Meo-PEG-b-PPMEMA polymer and a cationic lipid-like compound G0-C14 for in vivo delivery of cytotoxic saporin and breast cancer therapy. This nanoplatform could respond to a TME pH to rapidly release saporin/G0-C14 complexes, which could significantly improve the uptake of cytosolic saporin by tumor cells and subsequent endosomal escape, thereby leading to an effective inhibition of tumor growth.
Asunto(s)
Antineoplásicos/química , Neoplasias de la Mama/tratamiento farmacológico , Nanocápsulas/química , Polímeros/química , Saporinas/química , Microambiente Tumoral/fisiología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Composición de Medicamentos , Liberación de Fármacos , Femenino , Humanos , Concentración de Iones de Hidrógeno , Ratones , Neoplasias Experimentales , Saporinas/farmacología , Factores de TiempoRESUMEN
Saporin, a type I ribosome-inactivating protein from soapwort plant, is a potent protein synthesis inhibitor. Catalytically, saporin is a characteristic N-glycosidase, and it depurinates a specific adenine residue from a universally conserved loop of the major ribosomal RNA (rRNA) of eukaryotic cells. It is well-known that saporin induces apoptosis through different pathways, including ribotoxic stress response, cell signal transduction, genomic DNA fragmentation and RNA abasic lyase (RAlyase) activity, and NAD+ depletion by poly-(ADP)-ribose polymerase hyperactivation. Saporin's high enzymatic activity, high stability, and resistance to conjugation procedures make it a well-suited tool for immunotherapy approaches.In the present study, we focus on saporin-based targeted toxins that may be efficacious therapeutic agents for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our discussed points suggest that saporin may be a strategic molecule for therapeutic knockout treatments and a powerful candidate for novel drugs in the struggle against coronavirus 2019 (COVID-19).
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Saporinas/química , Saporinas/farmacología , Antivirales/química , Apoptosis/efectos de los fármacos , Humanos , Inmunotoxinas/química , Inmunotoxinas/farmacología , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Motoneuron loss is a severe medical problem that can result in loss of motor control and eventually death. We have previously demonstrated that partial motoneuron loss can result in dendritic atrophy and functional deficits in nearby surviving motoneurons, and that an androgen-dependent effect of exercise following injury can be neuroprotective against this dendritic atrophy. In this study, we explored where the necessary site of androgen action is for exercise-driven neuroprotective effects on induced dendritic atrophy. Motoneurons innervating the vastus medialis muscles of adult male rats were selectively killed by intramuscular injection of cholera toxin-conjugated saporin. Simultaneously, some saporin-injected animals were given implants of the androgen receptor antagonist hydroxyflutamide, either directly at the adjacent vastus lateralis musculature ipsilateral to the saporin-injected vastus medialis or interscapularly as a systemic control. Following saporin injections, some animals were allowed free access to a running wheel attached to their home cages. Four weeks later, motoneurons innervating the same vastus lateralis muscle were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Dendritic arbor lengths of saporin-injected animals allowed to exercise were significantly longer than those not allowed to exercise. Androgen receptor blockade locally at the vastus lateralis muscle prevented the protective effect of exercise. These findings indicate that exercise following neural injury exerts a protective effect on motoneuron dendrites, which acts via androgen receptor action at the target muscle.
Asunto(s)
Andrógenos , Atrofia/patología , Peroxidasa de Rábano Silvestre/metabolismo , Receptores Androgénicos , Saporinas/química , Médula Espinal/fisiología , Animales , Toxina del Cólera , Dendritas/patología , Peroxidasa de Rábano Silvestre/química , Masculino , Neuronas Motoras/patología , Músculo Esquelético/patología , Ratas , Ratas Sprague-Dawley , Saporinas/metabolismoRESUMEN
Cholesterol seems to play a central role in the augmentation of saporin-based immunotoxin (IT) cytotoxicity by triterpenoid saponins. Endolysosomal escape has been proposed as one mechanism for the saponin-mediated enhancement of targeted toxins. We investigated the effects of lipid depletion followed by repletion on Saponinum album (SA)-induced endolysosomal escape of Alexa Fluor labelled saporin and the saporin-based immunotoxin OKT10-SAP, directed against CD38, in Daudi lymphoma cells. Lipid deprived cells showed reduced SA-induced endolysosomal escape at two concentrations of SA, as determined by a flow cytometric method. The repletion of membrane cholesterol by low density lipoprotein (LDL) restored SA-induced endolysosomal escape at a concentration of 5 µg/mL SA but not at 1 µg/mL SA. When LDL was used to restore the cholesterol levels in lipid deprived cells, the SA augmentation of OKT10-SAP cytotoxicity was partially restored at 1 µg/mL SA and fully restored at 5 µg/mL SA. These results suggest that different mechanisms of action might be involved for the two different concentrations of SA and that endosomal escape may not be the main mechanism for the augmentation of saporin IT cytotoxicity by SA at the sub-lytic concentration of 1 µg/mL SA.
Asunto(s)
Colesterol/química , Endosomas/efectos de los fármacos , Inmunotoxinas/metabolismo , Lisosomas/efectos de los fármacos , Saponinas/farmacología , Saporinas/metabolismo , ADP-Ribosil Ciclasa 1/metabolismo , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Colesterol/metabolismo , LDL-Colesterol/farmacología , Relación Dosis-Respuesta a Droga , Endosomas/química , Endosomas/metabolismo , Fluoresceínas/química , Colorantes Fluorescentes/química , Humanos , Inmunotoxinas/química , Linfocitos/química , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Lisosomas/química , Lisosomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Saporinas/química , Ácidos Sulfónicos/química , Triterpenos/farmacologíaRESUMEN
Antibody-Drug Conjugates (ADCs) developed as a targeted treatment approach to deliver toxins directly to cancer cells are one of the fastest growing classes of oncology therapeutics, with eight ADCs and two immunotoxins approved for clinical use. However, selection of an optimum target and payload combination, to achieve maximal therapeutic efficacy without excessive toxicity, presents a significant challenge. We have developed a platform to facilitate rapid and cost-effective screening of antibody and toxin combinations for activity and safety, based on streptavidin-biotin conjugation. For antibody selection, we evaluated internalization by target cells using streptavidin-linked antibodies conjugated to biotinylated saporin, a toxin unable to cross cell membranes. For payload selection, we biotinylated toxins and conjugated them to antibodies linked to streptavidin to evaluate antitumour activity and pre-clinical safety. As proof of principle, we compared trastuzumab conjugated to emtansine via streptavidin-biotin (Trastuzumab-SB-DM1) to the clinically approved trastuzumab emtansine (T-DM1). We showed comparable potency in reduction of breast cancer cell survival in vitro and in growth restriction of orthotopic breast cancer xenografts in vivo. Our findings indicate efficient generation of functionally active ADCs. This approach can facilitate the study of antibody and payload combinations for selection of promising candidates for future ADC development.
Asunto(s)
Antineoplásicos/química , Inmunoconjugados/química , Toxinas Biológicas/química , Trastuzumab/química , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biotina/química , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Maitansina/química , Ratones , Ratones Endogámicos NOD , Ratones SCID , Saporinas/química , Estreptavidina/química , Trasplante Heterólogo , Trastuzumab/uso terapéuticoRESUMEN
Rice is the most important staple food in the world, but rice production is challenged by several biotic stress factors like viruses, bacteria, fungi and pest insects. One of the most notorious pest insects is Nilaparvata lugens, commonly known as the brown planthopper, which feeds on rice phloem sap and can cause serious damage to rice fields. In order to protect themselves, plants express a wide array of defense proteins such as ribosome-inactivating proteins (RIPs). This study shows that the expression of 'OsRIP1' is highly induced in rice plants infested with N. lugens, with transcript levels more than 100-fold upregulated in infested plants compared to non-infested plants. Furthermore, recombinant OsRIP1 was toxic for brown planthoppers when administered through liquid artificial diet. OsRIP1 inactivated insect ribosomes in vitro, suggesting that its toxicity relates to the enzymatic activity of OsRIP1. Over-expression of OsRIP1 in transgenic rice plants did not affect the performance of insects reared on these plants, most likely due to insufficient concentrations of OsRIP1 in the phloem. The data obtained in this research indicate that OsRIP1 can play a role in plant defense against herbivorous insects.
Asunto(s)
Hemípteros/efectos de los fármacos , Oryza/química , Fitoquímicos/farmacología , Saporinas/metabolismo , Animales , Fitoquímicos/química , Fitoquímicos/aislamiento & purificación , Saporinas/química , Saporinas/aislamiento & purificaciónRESUMEN
Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein involved in cell proliferation and differentiation. Ribosomal inactivating proteins derived from plants specifically target ribosomes and irreversibly inhibit protein synthesis. EpCAM antibody and saporin were conjugated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide chemistry. The mass of the conjugates were characterised using matrix-assisted laser desorption ionisation (MALDI). The saporin-EpCAM (SAP-EpAB) conjugates were tested in-vitro against MCF-7 (breast cancer cells), WERI-Rb1 (retinoblastoma) cells. The flow cytometry and fluorescence microscopy were performed to show the binding efficiency of SAP-EpAB conjugate. Whole transcriptome changes of sap-conjugate treated cells were studied using affymetrix microarrays. MALDI-TOF analysis and polyacrylamide gel electrophoresis confirmed the conjugation of SAP with EpCAM antibody. Flow cytometry and fluorescent microscopy analysis revealed the binding of SAP-EpAB conjugates to the MCF-7, WERI-Rb1 cells. Apoptosis assay by annexin-V has shown an increased apoptotic and necrotic population in conjugate treated cells. MTT assay confirmed the tumour cell death and had shown the IC50 value of 0.8â µg for conjugate in MCF-7 (breast cancer cells), and 1â µg for WERI-Rb1 (retinoblastoma) cells. The microarray analysis revealed downregulation of the tumourigenic genes and upregulation of pro-apoptotic genes leading to apoptosis of tumour cells.
Asunto(s)
Anticuerpos/metabolismo , Antineoplásicos/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Molécula de Adhesión Celular Epitelial/metabolismo , Saporinas/química , Anticuerpos/química , Anticuerpos/inmunología , Anticuerpos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Molécula de Adhesión Celular Epitelial/inmunología , Humanos , Células MCF-7 , Saporinas/metabolismoRESUMEN
In cancer treatment, polymeric nanoparticles (NPs) can serve as a vehicle for the delivery of cytotoxic proteins that have intracellular targets but that lack well-defined mechanisms for cellular internalization, such as saporin. In this work, we have prepared PEGylated poly(lactic acid- co-glycolic acid- co-hydroxymethyl glycolic acid) (PLGHMGA) NPs for the selective delivery of saporin in the cytosol of HER2 positive cancer cells. This selective uptake was achieved by decorating the surface of the NPs with the 11A4 nanobody that is specific for the HER2 receptor. Confocal microscopy observations showed rapid and extensive uptake of the targeted NPs (11A4-NPs) by HER2 positive cells (SkBr3) but not by HER2 negative cells (MDA-MB-231). This selective uptake was blocked upon preincubation of the cells with an excess of nanobody. Nontargeted NPs (Cys-NPs) were not taken up by either type of cells. Importantly, a dose-dependent cytotoxic effect was only observed on SkBr3 cells when these were treated with saporin-loaded 11A4-NPs in combination with photochemical internalization (PCI), a technique that uses a photosensitizer and local light exposure to facilitate endosomal escape of entrapped nanocarriers and biomolecules. The combined use of saporin-loaded 11A4-NPs and PCI strongly inhibited cell proliferation and decreased cell viability through induction of apoptosis. Also the cytotoxic effect could be reduced by an excess of nanobody, reinforcing the selectivity of this system. These results suggest that the combination of the targeting nanobody on the NPs with PCI are effective means to achieve selective uptake and cytotoxicity of saporin-loaded NPs.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Nanopartículas/administración & dosificación , Polímeros/química , Receptor ErbB-2/metabolismo , Saporinas/administración & dosificación , Anticuerpos de Dominio Único/administración & dosificación , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Nanopartículas/química , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Poliésteres/química , Saporinas/química , Anticuerpos de Dominio Único/inmunología , Células Tumorales CultivadasRESUMEN
Ribosome-inactivating proteins (RIPs) are enzymes with N-glycosylase activity that remove adenine bases from the ribosomal RNA. In theory, one single RIP molecule internalized into a cell is sufficient to induce cell death. For this reason, RIPs are of high potential as toxic payload for anti-tumor therapy. A considerable number of RIPs are synthesized by plants that belong to the carnation family (Caryophyllaceae). Prominent examples are the RIPs saporin from Saponaria officinalis L. or dianthin from Dianthus caryophyllus L. In this study, we have isolated and characterized a novel RIP (termed gypsophilin-S) from the tiny seeds of Gypsophila elegans M. Bieb. (Caryophyllaceae). It is noteworthy that this is the first study presenting the complete amino acid sequence of a RIP from a Gypsophila species. Gypsophilin-S was isolated from the defatted seed material following ammonium sulphate precipitation and HPLC-based ion exchange chromatography. Gypsophilin-S-containing fractions were analysed by SDS-PAGE and mass spectrometry. The full amino acid sequence of gypsophilin-S was assembled by MALDI-TOF-MS-MS and PCR. Gypsophilin-S exhibited strong adenine releasing activity and its cytotoxicity in human glioblastoma cells was investigated using an impedance-based real-time assay in comparison with recombinant saporin and dianthin.
Asunto(s)
Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Caryophyllaceae/enzimología , Saporinas/química , Saporinas/farmacología , Semillas/enzimología , Secuencia de Aminoácidos , Antineoplásicos Fitogénicos/aislamiento & purificación , Línea Celular Tumoral , Humanos , Modelos Moleculares , Conformación Proteica , Saporinas/aislamiento & purificaciónRESUMEN
Endosomal entrapment is a key issue for the intracellular delivery of many nano-sized biotherapeutics to their cytosolic or nuclear targets. Photochemical internalisation (PCI) is a novel light-based solution that can be used to trigger the endosomal escape of a range of bioactive agents into the cytosol leading to improved efficacy in pre-clinical and clinical studies. PCI typically depends upon the endolysosomal colocalisation of the bioactive agent with a suitable photosensitiser that is administered separately. In this study we demonstrate that both these components may be combined for codelivery via a novel multifunctional liposomal nanocarrier, with a corresponding increase in the biological efficacy of the encapsulated agent. As proof of concept, we show here that the cytotoxicity of the 30 kDa protein toxin, saporin, in MC28 fibrosarcoma cells is significantly enhanced when delivered via a cell penetrating peptide (CPP)-modified liposome, with the CPP additionally functionalised with a photosensitiser that is targeted to endolysosomal membranes. This innovation opens the way for the efficient delivery of a range of biotherapeutics by the PCI approach, incorporating a clinically proven liposome delivery platform and using bioorthogonal ligation chemistries to append photosensitisers and peptides of choice.
Asunto(s)
Citosol/metabolismo , Citotoxinas/metabolismo , Liposomas/química , Fármacos Fotosensibilizantes/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Citotoxinas/química , Citotoxinas/farmacología , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Luz , Liposomas/síntesis química , Maleimidas/química , Microscopía Confocal , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Porfirinas/química , Porfirinas/metabolismo , Ratas , Saporinas/química , Saporinas/metabolismoRESUMEN
Plant Ribosome-inactivating proteins (RIPs) including the type I RIP Saporin have been used for the construction of Immunotoxins (ITxs) obtained via chemical conjugation of the toxic domain to whole antibodies or by generating genetic fusions to antibody fragments/targeting domains able to direct the chimeric toxin against a desired sub-population of cancer cells. The high enzymatic activity, stability and resistance to conjugation procedures and especially the possibility to express recombinant fusions in yeast, make Saporin a well-suited tool for anti-cancer therapy approaches. Previous clinical work on RIPs-based Immunotoxins (including Saporin) has shown that several critical issues must be taken into deeper consideration to fully exploit their therapeutic potential. This review focuses on possible combinatorial strategies (chemical and genetic) to augment Saporin-targeted toxin efficacy. Combinatorial approaches may facilitate RIP escape into the cytosolic compartment (where target ribosomes are), while genetic manipulations may minimize potential adverse effects such as vascular-leak syndrome or may identify T/B cell epitopes in order to decrease the immunogenicity following similar strategies as those used in the case of bacterial toxins such as Pseudomonas Exotoxin A or as for Type I RIP Bouganin. This review will further focus on strategies to improve recombinant production of Saporin-based chimeric toxins.
Asunto(s)
Inmunotoxinas , Saporinas , Animales , Terapia Genética , Humanos , Inmunotoxinas/química , Inmunotoxinas/genética , Inmunotoxinas/uso terapéutico , Fototerapia , Pinocitosis , Saporinas/química , Saporinas/genética , Saporinas/uso terapéuticoRESUMEN
Targeted anticancer therapies demand discovery of new cellular targets to be exploited for the delivery of toxic molecules and drugs. In this perspective, in the last few years, nucleolin has been identified as an interesting surface marker to be used for the therapy of glioblastoma. In this study, we investigated whether a synthetic antagonist of cell-surface nucleolin known as N6L, previously reported to decrease both tumor growth and tumor angiogenesis in several cancer cell lines, including glioblastoma cells, as well as endothelial cells proliferation, could be exploited to deliver a protein toxin (saporin) to glioblastoma cells. The pseudopeptide N6L cross-linked to saporin-S6 induced internalization of the toxin inside glioblastoma cancer cells. Our results in vitro demonstrated the effectiveness of this conjugate in inducing cell death, with an ID50 four orders of magnitude lower than that observed for free N6L. Furthermore, the preliminary in vivo study demonstrated efficiency in reducing the tumor mass in an orthotopic mouse model of glioblastoma.