RESUMEN
The susceptibility of lysosomal membranes in tumor cells to cationic amphiphilic drugs (CADs) enables CADs to induce lysosomal membrane permeabilization (LMP) and trigger lysosome-dependent cell death (LDCD), suggesting a potential antitumor therapeutic approach. However, the existence of intrinsic lysosomal damage response mechanisms limits the display of the pharmacological activity of CADs. In this study, we report that low concentrations of QS-21, a saponin with cationic amphiphilicity extracted from Quillaja Saponaria tree, can induce LMP but has nontoxicity to tumor cells. QS-21 and MAP30, a type I ribosome-inactivating protein, synergistically induce apoptosis in tumor cells at low concentrations of both. Mechanistically, QS-21-induced LMP helps MAP30 escape from endosomes or lysosomes and subsequently enter the endoplasmic reticulum, where MAP30 downregulates the expression of autophagy-associated LC3 proteins, thereby inhibiting lysophagy. The inhibition of lysophagy results in the impaired clearance of damaged lysosomes, leading to the leakage of massive lysosomal contents such as cathepsins into the cytoplasm, ultimately triggering LDCD. In summary, our study showed that coadministration of QS-21 and MAP30 amplified the lysosomal disruption and can be a new synergistic LDCD-based antitumor therapy.
Asunto(s)
Antineoplásicos , Apoptosis , Autofagia , Lisosomas , Proteínas Inactivadoras de Ribosomas Tipo 1 , Saponinas , Animales , Humanos , Ratones , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Sinergismo Farmacológico , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Quillaja/química , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Saponinas/farmacologíaRESUMEN
Glucoprivic feeding is one of several counterregulatory responses (CRRs) that facilitates restoration of euglycemia following acute glucose deficit (glucoprivation). Our previous work established that glucoprivic feeding requires ventrolateral medullary (VLM) catecholamine (CA) neurons that coexpress neuropeptide Y (NPY). However, the connections by which VLM CA/NPY neurons trigger increased feeding are uncertain. We have previously shown that glucoprivation, induced by an anti-glycolygic agent 2-deoxy-D-glucose (2DG), activates perifornical lateral hypothalamus (PeFLH) neurons and that expression of NPY in the VLM CA/NPY neurons is required for glucoprivic feeding. We therefore hypothesized that glucoprivic feeding and possibly other CRRs require NPY-sensitive PeFLH neurons. To test this, we used the ribosomal toxin conjugate NPY-saporin (NPY-SAP) to selectively lesion NPY receptor-expressing neurons in the PeFLH of male rats. We found that NPY-SAP destroyed a significant number of PeFLH neurons, including those expressing orexin, but not those expressing melanin-concentrating hormone. The PeFLH NPY-SAP lesions attenuated 2DG-induced feeding but did not affect 2DG-induced increase in locomotor activity, sympathoadrenal hyperglycemia, or corticosterone release. The 2DG-induced feeding response was also significantly attenuated in NPY-SAP-treated female rats. Interestingly, PeFLH NPY-SAP lesioned male rats had reduced body weights and decreased dark cycle feeding, but this effect was not seen in female rats. We conclude that a NPY projection to the PeFLH is necessary for glucoprivic feeding, but not locomotor activity, hyperglycemia, or corticosterone release, in both male and female rats.
Asunto(s)
Conducta Alimentaria , Hipotálamo , Neuronas , Neuropéptido Y , Ratas Sprague-Dawley , Animales , Femenino , Masculino , Ratas , Desoxiglucosa/farmacología , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Alimentos/fisiología , Conducta Alimentaria/efectos de los fármacos , Glucosa/metabolismo , Área Hipotalámica Lateral/metabolismo , Área Hipotalámica Lateral/efectos de los fármacos , Hormonas Hipotalámicas/metabolismo , Hipotálamo/metabolismo , Hipotálamo/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Melaninas/metabolismo , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Neuropéptido Y/metabolismo , Neuropéptido Y/farmacología , Neuropéptidos/metabolismo , Orexinas/metabolismo , Hormonas Hipofisarias/metabolismo , Receptores de Neuropéptido Y/metabolismo , Receptores de Neuropéptido Y/genética , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Saporinas/farmacologíaRESUMEN
Among all kinds of anticancer agents, small molecule drugs produce an unsatisfactory therapeutic effect due to the lack of selectivity, notorious drug resistance and side effects. Therefore, researchers have begun to pay extensive attention to macromolecular drugs with high efficacy and specificity. As a plant toxin, gelonin exerts potent antitumor activity via inhibiting intracellular protein synthesis. However, gelonin lacks a translocation domain, and thus its poor cellular uptake leads to low outcomes of antitumor response. Here, tumor acidity and matrix metalloproteinase (MMP) dual-responsive functional gelonin (Trx-PVGLIG-pHLIP-gelonin, TPpG), composed of a thioredoxin (Trx) tag, a pH low insertion peptide (pHLIP), an MMP-responsive motif PVGLIG hexapeptide and gelonin, was innovatively proposed and biologically synthesized by a gene recombination technique. TPpG exhibited good thermal and serum stability, showed MMP responsiveness and could enter tumor cells under weakly acidic conditions, especially for MMP2-overexpressing HT1080 cells. Compared to low MMP2-expressing MCF-7 cells, TPpG displayed enhanced in vitro antitumor efficacy to HT1080 cells at pH 6.5 as determined by different methods. Likewise, TPpG was much more effective in triggering cell apoptosis and inhibiting protein synthesis in HT1080 cells than in MCF-7 cells. Intriguingly, with enhanced stability and pH/MMP dual responsiveness, TPpG notably inhibited subcutaneous HT1080 xenograft growth in mice and no noticeable off-target side effect was observed. This ingeniously designed strategy aims at providing new perspectives for the development of a smart platform that can intelligently respond to a tumor microenvironment for efficient protein delivery.
Asunto(s)
Antineoplásicos , Neoplasias , Humanos , Ratones , Animales , Metaloproteinasa 2 de la Matriz , Proteínas Inactivadoras de Ribosomas Tipo 1/química , Proteínas Inactivadoras de Ribosomas Tipo 1/genética , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Células MCF-7 , Neoplasias/tratamiento farmacológicoRESUMEN
The ventral pallidum (VP), a major component of the reward circuit, is well-associated with appetitive behaviors. Recent evidence suggests that this basal forebrain nucleus may have an overarching role in affective processing, including behavioral responses to aversive stimuli. We investigated this by utilizing selective immunotoxin lesions and a series of behavioral tests in adult male Wistar rats. We made bilateral GAT1-Saporin, 192-IgG-Saporin or PBS (vehicle) injections into the VP to respectively eliminate GABAergic and cholinergic neurons, and tested the animals in the forced swim test (FST), open field test (OFT), elevated plus maze (EPM), Morris water maze (MWM) and cued fear conditioning. Both GAT1-Saporin and 192-IgG-Saporin injections reduced behavioral despair without altering general locomotor activity. During the acquisition phase of cued fear conditioning, this antidepressant effect was accompanied by reduced freezing and increased darting in the 192-IgG-Saporin group, and increased jumping in the GAT1-Saporin group. In the extinction phase, cholinergic lesions impaired fear memory irrespective of the context, while GABAergic lesions reduced memory durability only during the early phases of extinction in a novel context. In line with this, selective cholinergic, but not GABAergic, lesions impaired spatial memory in the MWM. We observed no consistent effect in anxiety-like behavior assessed in the OFT and EPM. These findings indicate that both the GABAergic and cholinergic neuronal groups of the VP may contribute to emotion regulation through modulation of behavioral despair and acquired fear by suppressing active coping and promoting species-specific passive behaviors.
Asunto(s)
Prosencéfalo Basal , Ratas , Animales , Masculino , Saporinas , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Aprendizaje por Laberinto , Ratas Wistar , Neuronas Colinérgicas/fisiología , Colinérgicos/farmacología , Trastornos de la Memoria , Miedo , Adaptación Psicológica , Inmunoglobulina GRESUMEN
Curcin and Curcin C, both of the ribosome-inactivating proteins of Jatropha curcas, have apparent inhibitory effects on the proliferation of osteosarcoma cell line U20S. However, the inhibitory effect of the latter is 13-fold higher than that of Curcin. The mechanism responsible for the difference has not been studied. This work aimed to understand and verify whether there are differences in entry efficiency and pathway between them using specific endocytosis inhibitors, gene silencing, and labeling techniques such as fluorescein isothiocyanate (FITC) labeling. The study found that the internalization efficiency of Curcin C was twice that of Curcin for U2OS cells. More than one entering pathway was adopted by both of them. Curcin C can enter U2OS cells through clathrin-dependent endocytosis and macropinocytosis, but clathrin-dependent endocytosis was not an option for Curcin. The low-density lipoprotein receptor-related protein 1 (LRP1) was found to mediate clathrin-dependent endocytosis of Curcin C. After LRP1 silencing, there was no significant difference in the 50% inhibitory concentration (IC50) and endocytosis efficiency between Curcin and Curcin C on U2OS cells. These results indicate that LRP1-mediated endocytosis is specific to Curcin C, thus leading to higher U2OS endocytosis efficiency and cytotoxicity than Curcin.
Asunto(s)
Alcaloides , Jatropha , Osteosarcoma , Toxinas Biológicas , Humanos , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Jatropha/genética , Jatropha/metabolismo , Proteínas Inactivadoras de Ribosomas/metabolismo , Toxinas Biológicas/metabolismo , Alcaloides/metabolismo , Clatrina/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismoRESUMEN
Suicide gene therapy is a relatively novel form of cancer therapy in which a gene coding for enzymes or protein toxins is delivered through targeting systems such as vesicles, nanoparticles, peptide or lipidic co-adjuvants. The use of toxin genes is particularly interesting since their catalytic activity can induce cell death, damaging in most cases the translation machinery (ribosomes or protein factors involved in protein synthesis) of quiescent or proliferating cells. Thus, toxin gene delivery appears to be a promising tool in fighting cancer. In this review we will give an overview, describing some of the bacterial and plant enzymes studied so far for their delivery and controlled expression in tumor models.
Asunto(s)
Inmunotoxinas , Neoplasias , Toxinas Biológicas , Terapia Genética , Humanos , Inmunotoxinas/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Ribosomas/metabolismo , Toxinas Biológicas/metabolismoRESUMEN
Saporin is a ribosome-inactivating protein that can cause inhibition of protein synthesis and causes cell death when delivered inside a cell. Development of commercial Saporin results in a technology termed 'molecular surgery', with Saporin as the scalpel. Its low toxicity (it has no efficient method of cell entry) and sturdy structure make Saporin a safe and simple molecule for many purposes. The most popular applications use experimental molecules that deliver Saporin via an add-on targeting molecule. These add-ons come in several forms: peptides, protein ligands, antibodies, even DNA fragments that mimic cell-binding ligands. Cells that do not express the targeted cell surface marker will not be affected. This review will highlight some newer efforts and discuss significant and unexpected impacts on science that molecular surgery has yielded over the last almost four decades. There are remarkable changes in fields such as the Neurosciences with models for Alzheimer's Disease and epilepsy, and game-changing effects in the study of pain and itch. Many other uses are also discussed to record the wide-reaching impact of Saporin in research and drug development.
Asunto(s)
Disciplinas de las Ciencias Biológicas , Inmunotoxinas , Indicadores y Reactivos , Ligandos , N-Glicosil Hidrolasas , Proteínas de Plantas/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , SaporinasRESUMEN
Osteosarcoma is a kind of primary bone malignant tumors. Its cure rate has been stagnant in the past decade years. Curcin C belongs to type I ribosome inactivating proteins, extracted from the cotyledons of post-germinated Jatropha curcas seeds. It can inhibit the proliferation of several tumor lines including U2OS cells with extraordinary efficiency. The treated U2OS cells were arrested in both S and G2/M phase, showed typical apoptosis morphological characteristic, formed autophagosomes and increase the ratio of LC3II to LC3I. Meanwhile, the level of ROS in the treated cells was found increasing significantly, with the change of mitochondrial membrane potential and decreased antioxidant enzyme activities. The application of ROS scavenger NAC not only significantly inhibited the toxicity of Curcin C but also prevented the happen of apoptosis and autophagy to some extent. These results suggested that Curcin C may function through ROS pathway. In addition, the Curcin C treatment could activate JNK and inhibit ERK signal pathway. Sp600125, an inhibitor of JNK signaling pathway, can prevent subsequent apoptosis and autophagy events, suggesting that JNK pathway was at least one of the pathways of Curcin C action. Moreover, the relevant including antagonistic among autophagy, apoptosis and cell cycle arresting induced by Curcin C also was found. In summary, it can be speculated that Curcin C may induce S, G2/M phase arrest, apoptosis and autophagy of human osteosarcoma U2OS cells through activating JNK signal pathway and blocking ERK signal pathway by promoting ROS accumulation in cell, thus finally reflected in the effect of inhibiting tumor cell proliferation.
Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Antioxidantes/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Proteínas Inactivadoras de Ribosomas Tipo 1/química , Proteínas Inactivadoras de Ribosomas Tipo 1/aislamiento & purificaciónRESUMEN
Using the pathosystem Phaseolus vulgaris-tobacco necrosis virus (TNV), we demonstrated that PD-L1 and PD-L4, type-1 ribosome inactivating proteins (RIPs) from leaves of Phytolacca dioica L., possess a strong antiviral activity. This activity was exerted both when the RIPs and the virus were inoculated together in the same leaf and when they were inoculated or applied separately in the adaxial and abaxial leaf surfaces. This suggests that virus inhibition would mainly occur inside plant cells at the onset of infection. Histochemical studies showed that both PD-L1 and PD-L4 were not able to induce oxidative burst and cell death in treated leaves, which were instead elicited by inoculation of the virus alone. Furthermore, when RIPs and TNV were inoculated together, no sign of H2O2 deposits and cell death were detectable, indicating that the virus could have been inactivated in a very early stage of infection, before the elicitation of a hypersensitivity reaction. In conclusion, the strong antiviral activity is likely exerted inside host cells as soon the virus disassembles to start translation of the viral genome. This activity is likely directed towards both viral and ribosomal RNA, explaining the almost complete abolition of infection when virus and RIP enter together into the cells.
Asunto(s)
Antígeno B7-H1/farmacología , Phaseolus/virología , Phytolacca/química , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Tombusviridae/efectos de los fármacos , Antivirales/farmacología , Antígeno B7-H1/aislamiento & purificación , Interacciones Microbiota-Huesped , Hojas de la Planta/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1/aislamiento & purificaciónRESUMEN
Plant-based saponins are amphipathic glycosides composed of a hydrophobic aglycone backbone covalently bound to one or more hydrophilic sugar moieties. Recently, the endosomal escape activity of triterpenoid saponins has been investigated as a potentially powerful tool for improved cytosolic penetration of protein drugs internalized by endocytic uptake, thereby greatly enhancing their pharmacological effects. However, only a few saponins have been studied, and the paucity in understanding the structure-activity relationship of saponins imposes significant limitations on their applications. To address this knowledge gap, 12 triterpenoid saponins with diverse structural side chains were screened for their utility as endosomolytic agents. These compounds were used in combination with a toxin (MAP30-HBP) comprising a type I ribosome-inactivating protein fused to a cell-penetrating peptide. Suitability of saponins as endosomolytic agents was assessed on the basis of cytotoxicity, endosomal escape promotion, and synergistic effects on toxins. Five saponins showed strong endosomal escape activity, enhancing MAP30-HBP cytotoxicity by more than 106 to 109 folds. These saponins also enhanced the apoptotic effect of MAP30-HBP in a pH-dependent manner. Additionally, growth inhibition of MAP30-HBP-treated SMMC-7721 cells was greater than that of similarly treated HeLa cells, suggesting that saponin-mediated endosomolytic effect is likely to be cell-specific. Furthermore, the structural features and hydrophobicity of the sugar side chains were analyzed to draw correlations with endosomal escape activity and derive predictive rules, thus providing new insights into structure-activity relationships of saponins. This study revealed new saponins that can potentially be exploited as efficient cytosolic delivery reagents for improved therapeutic drug effects.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Endosomas/efectos de los fármacos , Saponinas/química , Saponinas/farmacología , Triterpenos/química , Triterpenos/farmacología , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , Sistemas de Liberación de Medicamentos/métodos , Liberación de Fármacos , Sinergismo Farmacológico , Glicosilación , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Extractos Vegetales/química , Extractos Vegetales/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1/química , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Relación Estructura-ActividadRESUMEN
In recent decades, immunotoxins have attracted significant attention in treatment of a wide range of diseases including cancers due to their natural origins and their role in blocking crucial pathways within the cells. Ribosome inactivating proteins (RIPs) are efficient molecules in blocking protein synthesis through interactions with ribosomal rRNA molecules. cDNA molecule encoding HER2 scFv antibody fragment originated from trastuzumab attached to the mature alpha luffin gene fragment was subcloned into pET28a expression vector and expressed in different E. coli expression hosts. Identity of the expressed recombinant protein was investigated through western blotting and the fusion protein was purified using Ni-NTA affinity chromatography. The biological activity (toxicity) of the protein was investigated on DNA and RNA samples. A 58 kDa protein was expressed in E. coli. The best protein expression level was achieved in 0.2 mM IPTG at 30 °C in TB medium using E. coli BL21 (DE3) host strain. The fusion protein showed RNase and DNA glycosylase activity on tested RNA and DNA samples. DNA glycosylase activity of the recombinant fusion protein showed that alpha luffin part of this protein is active in conjugation to the scFv molecule and the expressed protein can be further studied in targeted biological in vitro assays.
Asunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Inmunotoxinas/genética , Proteínas Inactivadoras de Ribosomas Tipo 1/genética , Anticuerpos de Cadena Única/genética , Trastuzumab/genética , Línea Celular , Vectores Genéticos/genética , Humanos , Inmunotoxinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Anticuerpos de Cadena Única/farmacología , Trastuzumab/farmacologíaRESUMEN
Since it was discovered as the first human tumor virus in 1964, Epstein-Barr Virus (EBV) is now implicated in several types of malignancies. Accordingly, certain aspects of EBV pathobiology have shown promise in anti-cancer research in developing virus-targeting methods for EBV-associated cancers. The unique role of EBV nuclear antigen 1 (EBNA1) in triggering episome-dependent functions has made it as the only latent gene to be expressed in most EBV+ neoplasms. Dimeric EBNA1 binds to the replication origin (oriP) to display its biological impact on EBV-driven cell transformation and maintenance. Hence, EBNA1/oriP has been made an ideal drug target site for anti-EBV protocol development. GAP31 protein was originally isolated from the seeds of an ancient medicinal plant Gelonium multiflorum. Although GAP31 has been shown to exhibit both anti-viral and anti-tumor activity, current understanding of the mechanistic picture underlying GAP31 functioning is not clear. Herein, we identify the EBNA1 DNA-binding domain as a core for GAP31 binding by performing affinity pulldown assays. Recombinant GAP31 (rGAP31) was shown to impair EBNA1-induced dimerization; consequently, it abrogated both EBNA1/oriP-mediated binding and transcription. Importantly, the therapeutic effects of GAP31 showed its capability to abrogate EBV-driven cell transformation and proliferation, and EBV-dependent tumorigenesis in xenograft animal models. Notably, the EBNA1 binding-mutant rGAP31R166A/R169A simply exhibits defective phenotypes in the above-mentioned studies. Our data suggest rGAP31 is a potential anti-viral drug which can be applied to the development of therapeutic strategies against EBV-related malignancies.
Asunto(s)
Antivirales/farmacología , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/efectos de los fármacos , Extractos Vegetales/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Replicación del ADN , Femenino , Ratones Endogámicos NOD , Ratones SCID , Plantas Medicinales/química , Origen de Réplica , Replicación Viral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cell surface molecules aberrantly expressed or overexpressed by myeloid leukemic cells represent potential disease-specific therapeutic targets for antibodies. MUC1 is a polymorphic glycoprotein, the cleavage of which yields two unequal chains: a large extracellular α subunit containing a tandem repeat array bound in a strong noncovalent interaction to a smaller ß subunit containing the transmembrane and cytoplasmic domains. Because the α-chain can be released from the cell-bound domains of MUC1, agents directed against the α-chain will not effectively target MUC1+ cells. The MUC1 SEA (a highly conserved protein module so called from its initial identification in a sea urchin sperm protein, in enterokinase, and in agrin) domain formed by the binding of the α and ß chains represents a stable structure fixed to the cell surface at all times. DMB-5F3, a partially humanized murine anti-MUC1 SEA domain monoclonal antibody, was used to examine MUC1 expression in acute myeloid leukemia (AML) and was found to bind acute myelomonocytic and monocytic leukemia (AML-M4 and AML-M5) cell lines. We also examined monocytic neoplasms freshly obtained from patients including chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia, which were found to uniformly express MUC1. CD34+/lin-/CD38- or CD38+ presumed leukemic stem cell populations from CD34+ AML and CD34-CD38- or CD38+ populations from CD34- AML were also found to express MUC1, although at low percentages. Based on these studies, we generated an anti-MUC1 immunotoxin to directly gauge the cytotoxic efficacy of targeting AML-bound MUC1. Using single-chain DMB-5F3 fused to recombinant gelonin toxin, the degree of AML cytotoxicity was found to correlate with MUC1 expression. Our data support the use of an anti-MUC1 SEA module-drug conjugates to selectively target and inhibit MUC1-expressing myelomonocytic leukemic cells.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Sistemas de Liberación de Medicamentos , Inmunotoxinas/farmacología , Leucemia Mielomonocítica Crónica , Leucemia Mielomonocítica Juvenil , Mucina-1/inmunología , Proteínas de Neoplasias/inmunología , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Anticuerpos de Cadena Única/farmacología , Animales , Femenino , Humanos , Células K562 , Leucemia Mielomonocítica Crónica/tratamiento farmacológico , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Crónica/inmunología , Leucemia Mielomonocítica Crónica/patología , Leucemia Mielomonocítica Juvenil/tratamiento farmacológico , Leucemia Mielomonocítica Juvenil/genética , Leucemia Mielomonocítica Juvenil/inmunología , Leucemia Mielomonocítica Juvenil/patología , Masculino , Ratones , Mucina-1/genética , Proteínas de Neoplasias/genética , Células Madre NeoplásicasRESUMEN
Ribosome inactivating protein (RIP) catalyzes the cleavage of glycosidic bond formed between adenine and ribose sugar of ribosomal RNA to inactivate ribosomes. Previous structural studies have shown that RNA bases, adenine, guanine, and cytosine tend to bind to RIP in the substrate binding site. However, the mode of binding of uracil with RIP was not yet known. Here, we report crystal structures of two complexes of type 1 RIP from Momordica balsamina (MbRIP1) with base, uracil and nucleoside, uridine. The binding studies of MbRIP1 with uracil and uridine as estimated using fluorescence spectroscopy showed that the equilibrium dissociation constants (KD ) were 1.2 × 10-6 M and 1.4 × 10-7 M respectively. The corresponding values obtained using surface plasmon resonance (SPR) were found to be 1.4 × 10-6 M and 1.1 × 10-7 M, respectively. Structures of the complexes of MbRIP1 with uracil (Structure-1) and uridine (Structure-2) were determined at 1.70 and 1.98 Å resolutions respectively. Structure-1 showed that uracil bound to MbRIP1 at the substrate binding site but its mode of binding was significantly different from those of adenine, guanine and cytosine. However, the mode of binding of uridine was found to be similar to those of cytidine. As a result of binding of uracil to MbRIP1 at the substrate binding site, three water molecules were expelled while eight water molecules were expelled when uridine bound to MbRIP1.
Asunto(s)
Momordica/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1/metabolismo , Uracilo/química , Uridina/química , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/farmacología , Unión Proteica , Conformación Proteica , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1/química , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Ribosomas/química , Ribosomas/metabolismo , Resonancia por Plasmón de Superficie , Uracilo/metabolismo , Uracilo/farmacología , Uridina/metabolismo , Uridina/farmacologíaRESUMEN
Despite substantial advances in its treatment, brain cancer remains a life-threatening disease with a poor survival rate. The main challenges for the conventional chemotherapy include an insufficient efficacy of drugs and toxicity caused by their nonselective mode of action. Recently, great attention has been paid to highly potent macromolecules such as gelonin, a type 1 ribosome-inactivating protein that inhibits protein translation. However, gelonin is poorly internalised into tumour cells and cannot distinguish between cancer and normal cells. To overcome these challenges, we engineered in this study a recombinant gelonin fusion protein with chlorotoxin, known as a brain cancer-homing peptide. The gelonin-chlorotoxin (Gel-CLTX) fusion chimera, produced in Escherichia coli, possessed an equipotent N-glycosidase activity with that of unmodified gelonin and, furthermore, could be selectively internalised into U-87 MG glioma cells over noncancerous glial cells. Consequently, Gel-CLTX displayed substantial inhibition of protein translation in U-87 MG cells, which eventually led to significantly augmented tumouricidal effects. When tested against xenograft tumour-bearing mice, Gel-CLTX showed higher tumour accumulation and inhibition of tumour growth than did gelonin, with a low systemic toxicity. Taken together, our results demonstrate the feasibility of using a fusion strategy for enhanced chemotherapy of brain tumours.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Proteínas Inactivadoras de Ribosomas Tipo 1/administración & dosificación , Venenos de Escorpión/administración & dosificación , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/toxicidad , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Ingeniería Genética , Glioblastoma/patología , Humanos , Masculino , Ratones , Ratones Desnudos , Ratas , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1/toxicidad , Venenos de Escorpión/farmacología , Venenos de Escorpión/toxicidad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Ricin A chain (RTA) and Pokeweed antiviral proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential production amenability and sub-toxic antiviral value of novel fusion proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral protein isoform 1 from seeds (RTA-PAPS1) was produced in an E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and protein synthesis inhibitory activity assayed by comparison to the production of a control protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. The fusion protein was further optimized using in silico tools, produced in an E. coli in vivo expression system, purified by a three-step process from soluble lysate and confirmed in a protein synthesis inhibition activity assay. RESULTS: Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded protein was bioactive with a 50% protein synthesis inhibitory concentration (IC50) of 0.06 nM (3.63 ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03 nM (1.82 ng/ml) and a therapeutic index of > 21,818 with noticeable steric hindrance. Results also showed that the optimized protein ricin A chain mutant-Pokeweed antiviral protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function on protein synthesis inhibition activity, with an IC50 of 0.03 nM (1.82 ng/ml), and with minimal, if any, steric hindrance. CONCLUSIONS: Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant gain of function on protein synthesis inhibition and anti-HBV activities in vitro with a high therapeutic index and, thus, merit further development as potential potent antiviral agents against chronic HBV infection to be used as a standalone or in combination with existent therapies.
Asunto(s)
Escherichia coli/genética , Proteínas Recombinantes de Fusión , Proteínas Inactivadoras de Ribosomas Tipo 1 , Ricina , Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Proteínas de Plantas/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1/biosíntesis , Proteínas Inactivadoras de Ribosomas Tipo 1/genética , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Ricina/biosíntesis , Ricina/genética , Ricina/farmacologíaRESUMEN
Cell targeting protein toxins have gained increasing interest for cancer therapy aimed at increasing the therapeutic window and reducing systemic toxicity. Because recombinant expression of immunotoxins consisting of a receptor-binding and a cell-killing moiety is hampered by their high toxicity in a eukaryotic production host, most applications rely on recombinant production of fusion proteins consisting of an antibody fragment and a protein toxin in bacterial hosts such as Escherichia coli ( E. coli). These fusions often lack beneficial properties of whole antibodies like extended serum half-life or efficient endocytic uptake via receptor clustering. Here, we describe the production of full-length antibody immunotoxins using self-splicing split inteins. To this end, the short (11 amino acids) N-terminal intein part of the artificially designed split intein M86, a derivative of the Ssp DnaB intein, was recombinantly fused to the heavy chain of trastuzumab, a human epidermal growth factor receptor 2 (HER2) receptor targeting antibody and to a nanobody-Fc fusion targeting the HER1 receptor, respectively. Both antibodies were produced in Expi293F cells. The longer C-terminal counterpart of the intein was genetically fused to the protein toxins gelonin or Pseudomonas Exotoxin A, respectively, and expressed in E. coli via fusion to maltose binding protein. Using optimized in vitro splicing conditions, we were able to generate a set of specific and potent immunotoxins with IC50 values in the mid- to subpicomolar range.
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ADP Ribosa Transferasas/genética , Toxinas Bacterianas/genética , Exotoxinas/genética , Inmunotoxinas/genética , Inteínas , Pseudomonas/genética , Receptor ErbB-2/antagonistas & inhibidores , Trastuzumab/genética , Factores de Virulencia/genética , ADP Ribosa Transferasas/farmacología , Animales , Antineoplásicos Inmunológicos/metabolismo , Antineoplásicos Inmunológicos/farmacología , Toxinas Bacterianas/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Células CHO , Línea Celular Tumoral , Cricetulus , Receptores ErbB/antagonistas & inhibidores , Escherichia coli/genética , Exotoxinas/farmacología , Femenino , Humanos , Inmunotoxinas/farmacología , Ingeniería de Proteínas , Empalme de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1/genética , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Trastuzumab/farmacología , Factores de Virulencia/farmacología , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Background/Aims: Pokeweed antiviral protein (PAP) has been reported to downregulate Wnt/Jnk pathway and attenuate liver fibrosis. This study was designed to intensively explore the mechanism of anti-fibrosis effect of PAP. Materials and Methods: Hepatic stellate cell (HSC) activation was induced by high concentration of glucose. Cell viability was detected at different time points after PAP treatment. Meanwhile, hepatic fibrosis models in mice were induced by CCl4 injection. In the end, liver pathology was observed and contents of alanine transaminase, aspartate transaminase, lactic dehydrogenase, hyaluronic acid (HA), and laminin (LN) in serum together with hydroxyproline (Hyp) in liver were measured. The mRNA and protein expressions of HK2, PFKP, PCK1, and FBP1 as well as Jnk expression in HSC-T6 cells and liver tissue were detected by qPCR and western-blot, respectively. Results: Compared with high glucose, PAP reduced viability and expressions of HK2, PFKP, α-SMA, and Col1A1, where as enhanced the expressions of PCK1 and FBP1 in HSC-T6 cells (P < 0.05) respectively. PAP attenuated liver pathology, improved liver function, and reduced collagen deposition in liver tissue compared with the model group (P < 0.05) respectively. Moreover, PAP reduced expressions of HK2, PFKP, α-SMA, and Col1A1 where as increased the expression of PCK1 and FBP1 in the liver of mice compared with the model group (P < 0.05) respectively. Most importantly, PAP reduced the phosphorylation of Jnk both in cells and liver tissue compared with the model group (P < 0.05) respectively. Conclusions: Our results demonstrated that PAP attenuated liver fibrosis by regulating Wnt/Jnk-mediated glucose metabolism. It provided us a new target for the treatment of liver fibrosis.
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Tetracloruro de Carbono/toxicidad , Glucosa/metabolismo , Cirrosis Hepática/tratamiento farmacológico , Proteínas Inactivadoras de Ribosomas Tipo 1/administración & dosificación , Animales , Línea Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/metabolismo , Pruebas de Función Hepática , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratas , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Antimicrobial peptides, also called Host Defence Peptides (HDPs), are effectors of innate immune response found in all living organisms. In a previous report, we have identified by chemical fragmentation, and characterized the first cryptic antimicrobial peptide in PD-L4, a type 1 ribosome inactivating protein (RIP) from leaves of Phytolacca dioica L. We applied a recently developed bioinformatic approach to a further member of the differently expressed pool of type 1 RIPs from P. dioica (PD-L1/2), and identified two novel putative cryptic HDPs in its N-terminal domain. These two peptides, here named IKY31 and IKY23, exhibit antibacterial activities against planktonic bacterial cells and, interestingly, significant anti-biofilm properties against two Gram-negative strains. Here, we describe that PD-L1/2 derived peptides are able to induce a strong dose-dependent reduction in biofilm biomass, affect biofilm thickness and, in the case of IKY31, interfere with cell-to-cell adhesion, likely by affecting biofilm structural components. In addition to these findings, we found that both PD-L1/2 derived peptides are able to assume stable helical conformations in the presence of membrane mimicking agents (SDS and TFE) and intriguingly beta structures when incubated with extracellular bacterial wall components (LPS and alginate). Overall, the data collected in this work provide further evidence of the importance of cryptic peptides derived from type 1 RIPs in host/pathogen interactions, especially under pathophysiological conditions induced by biofilm forming bacteria. This suggests a new possible role of RIPs as precursors of antimicrobial and anti-biofilm agents, likely released upon defensive proteolytic processes, which may be involved in plant homeostasis.
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Antiinfecciosos/farmacología , Biopelículas/efectos de los fármacos , Phytolacca/química , Proteínas de Plantas/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Biología Computacional , Lipopolisacáridos/metabolismo , Proteínas de Plantas/química , Estructura Secundaria de Proteína , Proteínas Inactivadoras de Ribosomas Tipo 1/químicaRESUMEN
BACKGROUND: Tumour endothelial marker 1 (TEM1/endosialin/CD248) is a tumour-restricted cell-surface protein expressed by human sarcomas. We previously developed a high-affinity human single-chain variable fragment (scFv)-Fc fusion protein (78Fc) against TEM1 and demonstrated its specific binding to human and mouse TEM1. PATIENT AND METHODS: Clinical sarcoma specimens were collected between 2000 and 2015 at the Hospital of the University of Pennsylvania, as approved by the institutional review board and processed by standard formalin-fixed paraffin embedded techniques. We analysed TEM1 expression in 19 human sarcoma subtypes (n = 203 specimens) and eight human sarcoma-cell lines. Near-infrared (NIR) imaging of tumour-bearing mice was used to validate 78Fc binding to TEM1+ sarcoma in vivo. Finally, we tested an immunotoxin conjugate of anti-TEM1 78Fc with saporin (78Fc-Sap) for its therapeutic efficacy against human sarcoma in vitro and in vivo. RESULTS: TEM1 expression was identified by immunohistochemistry in 96% of human sarcomas, of which 81% expressed TEM1 both on tumour cells and the tumour vasculature. NIR imaging revealed specific in vivo targeting of labelled 78Fc to TEM1+ sarcoma xenografts. Importantly, 78Fc-Sap was effective in killing in vitro TEM1+ sarcoma cells and eliminated human sarcoma xenografts without apparent toxicity in vivo. CONCLUSION: TEM1 is an important therapeutic target for human sarcoma, and the high-affinity TEM1-specific scFv fusion protein 78Fc is suitable for further clinical development for therapeutic applications in sarcoma.