ABSTRACT
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.
Subject(s)
Brain Neoplasms/drug therapy , Glioblastoma/drug therapy , Ribosome Inactivating Proteins, Type 1/administration & dosage , Scorpion Venoms/administration & dosage , Animals , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/toxicity , Brain Neoplasms/pathology , Cell Line, Tumor , Genetic Engineering , Glioblastoma/pathology , Humans , Male , Mice , Mice, Nude , Rats , Ribosome Inactivating Proteins, Type 1/pharmacology , Ribosome Inactivating Proteins, Type 1/toxicity , Scorpion Venoms/pharmacology , Scorpion Venoms/toxicity , Xenograft Model Antitumor AssaysABSTRACT
Interactions between stromal cells and tumor cells pay a major role in cancer growth and progression. This is reflected in the composition of anticancer drugs which includes compounds directed towards the immune system and tumor-vasculature in addition to drugs aimed at the cancer cells themselves. Drug-based treatment regimens are currently designed to include compounds targeting the tumor stroma in addition to the cancer cells. Treatment limiting adverse effects remains, however, one of the major challenges for drug-based therapy and novel tolerable treatment modalities with diverse high efficacy on both tumor cells and stroma is therefore of high interest. It was hypothesized that the vascular targeted fusion toxin VEGF121/rGel in combination with the intracellular drug delivery technology photochemical internalization (PCI) stimulate direct cancer parenchymal cell death in addition to inhibition of tumor perfusion, and that an immune mediated response is relevant for treatment outcome. The aim of the present study was therefore to elucidate the anticancer mechanisms of VEGF121/rGel-PCI. In contrast to VEGF121/rGel monotherapy, VEGF121/rGel-PCI was found to mediate its effect through VEGFR1 and VEGFR2, and a targeted treatment effect was shown on two VEGFR1 expressing cancer cell lines. A cancer parenchymal treatment effect was further indicated on H&E stains of CT26-CL25 and 4â¯T1 tumors. VEGF121/rGel-PCI was shown, by dynamic contrast enhanced MRI, to induce a sustained inhibition of tumor perfusion in both tumor models. A 50% complete remission (CR) of CT26.CL25 colon carcinoma allografts was found in immunocompetent mice while no CR was detected in CT26.CL25 bearing athymic mice. In conclusion, the present report indicate VEGF121/rGel -PCI as a treatment modality with multimodal tumor targeted efficacy that should be further developed towards clinical utilization.
Subject(s)
Antineoplastic Agents, Phytogenic/administration & dosage , Drug Delivery Systems , Neoplasms/drug therapy , Ribosome Inactivating Proteins, Type 1/administration & dosage , Vascular Endothelial Growth Factor A/administration & dosage , Animals , Cell Line, Tumor , Female , Light , Mice, Inbred BALB C , Mice, Nude , Neoplasms/metabolism , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
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.
Subject(s)
Carbon Tetrachloride/toxicity , Glucose/metabolism , Liver Cirrhosis/drug therapy , Ribosome Inactivating Proteins, Type 1/administration & dosage , Animals , Cell Line , Disease Models, Animal , Gene Expression Regulation/drug effects , Hepatic Stellate Cells/cytology , Hepatic Stellate Cells/drug effects , Hepatic Stellate Cells/metabolism , Liver Cirrhosis/chemically induced , Liver Cirrhosis/metabolism , Liver Function Tests , MAP Kinase Signaling System/drug effects , Mice , Rats , Ribosome Inactivating Proteins, Type 1/pharmacology , Wnt Signaling Pathway/drug effectsABSTRACT
With the high efficacy of protein-based therapeutics and plenty of intracellular drug targets, cytosolic protein delivery in a cell-specific manner has attracted considerable attention in the field of precision medicine. Herein, we present an intracellular protein delivery system based on a target-specific repebody and the translocation domain of Pseudomonas aeruginosa exotoxin A. The delivery platform was constructed by genetically fusing an EGFR-specific repebody as a targeting moiety to the translocation domain, while a protein cargo was fused to the C-terminal end of the delivery platform. The delivery platform was revealed to efficiently translocate a protein cargo to the cytosol in a target-specific manner. We demonstrate the utility and potential of the delivery platform by showing a remarkable tumor regression with negligible toxicity in a xenograft mice model when gelonin was used as the cytotoxic protein cargo. The present platform can find wide applications to the cell-selective cytosolic delivery of diverse proteins in many areas.
Subject(s)
ADP Ribose Transferases/chemistry , Antineoplastic Agents, Phytogenic/administration & dosage , Bacterial Toxins/chemistry , Drug Carriers/chemistry , Exotoxins/chemistry , Neoplasms/drug therapy , Ribosome Inactivating Proteins, Type 1/administration & dosage , Virulence Factors/chemistry , Animals , Antineoplastic Agents, Phytogenic/therapeutic use , Cell Line, Tumor , Drug Delivery Systems , Humans , Male , Mice , Mice, Inbred BALB C , Models, Molecular , Protein Domains , Ribosome Inactivating Proteins, Type 1/therapeutic use , Pseudomonas aeruginosa Exotoxin AABSTRACT
Owing to the extraordinary potency in inhibiting protein translation that could eventually lead to apoptosis of tumor cells, ribosome-inactivating proteins (RIPs) such as gelonin have been considered attractive drug candidates for cancer therapy. However, due to several critical obstacles (e.g., severe toxicity issues caused by a lack of selectivity in their mode of action and the low cytotoxicity via poor cellular uptake, etc.), clinical application of RIPs is yet far from being accomplished. To overcome these challenges, in the present study, we engineered gelonin fusion proteins with anti-insulin-like growth factor-1 receptor (IGF-1R) affibody ("IAFF") via the genetic recombinant method and the SpyCatcher/SpyTag-mediated conjugation method. To this end, recombinant gelonin-anti-IGF-1R affibody (rGel-IAFF), gelonin-SpyCatcher (Gel-SpyCatcher) and SpyTag-IAFF fusion proteins were produced from the E. coli expression system, and gelonin-IAFF conjugate was synthesized by mixing Gel-SpyCatcher and SpyTag-IAFF. After preparation of both rGel-IAFF and Gel-IAFF conjugate, their components' functionality was characterized in vitro. Our assay results confirmed that, while both Gel-IAFF and Gel-SpyCatcher retained equipotent N-glycosidase activity to that of gelonin, IAFF was able to selectively bind to IGF-1R overexpressed U87 MG brain cancer cells over low expression LNCaP cells. The results of cellular analyses showed that rGel-IAFF and Gel-IAFF conjugate both exhibited a greater cell uptake in the U87 MG cells than gelonin, but not in the LNCaP cells, yielding a significantly augmented cytotoxicity only in the U87 MG cells. Remarkably, rGel-IAFF and Gel-IAFF conjugate displayed 22- and 5.6-fold lower IC50 values (avg. IC50: 180 and 720 nM, respectively) than gelonin (avg. IC50: 4000 nM) in the U87 MG cells. Overall, the results of the present research demonstrated that fusion of gelonin with IAFF could provide an effective way to enhance the anti-tumor activity, while reducing the associated toxicity of gelonin.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Brain Neoplasms/drug therapy , Recombinant Fusion Proteins/chemistry , Ribosome Inactivating Proteins, Type 1/pharmacology , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/chemistry , Brain Neoplasms/pathology , Cell Line, Tumor , Humans , Inhibitory Concentration 50 , Peptides/chemistry , Receptor, IGF Type 1/metabolism , Ribosome Inactivating Proteins, Type 1/administration & dosage , Ribosome Inactivating Proteins, Type 1/chemistryABSTRACT
There is a lack of tools that selectively target vagal afferent neurons (VAN) innervating the gut. We use saporin (SAP), a potent neurotoxin, conjugated to the gastronintestinal (GI) hormone cholecystokinin (CCK-SAP) injected into the nodose ganglia (NG) of male Wistar rats to specifically ablate GI-VAN. We report that CCK-SAP ablates a subpopulation of VAN in culture. In vivo, CCK-SAP injection into the NG reduces VAN innervating the mucosal and muscular layers of the stomach and small intestine but not the colon, while leaving vagal efferent neurons intact. CCK-SAP abolishes feeding-induced c-Fos in the NTS, as well as satiation by CCK or glucagon like peptide-1 (GLP-1). CCK-SAP in the NG of mice also abolishes CCK-induced satiation. Therefore, we provide multiple lines of evidence that injection of CCK-SAP in NG is a novel selective vagal deafferentation technique of the upper GI tract that works in multiple vertebrate models. This method provides improved tissue specificity and superior separation of afferent and efferent signaling compared with vagotomy, capsaicin, and subdiaphragmatic deafferentation.NEW & NOTEWORTHY We develop a new method that allows targeted lesioning of vagal afferent neurons that innervate the upper GI tract while sparing vagal efferent neurons. This reliable approach provides superior tissue specificity and selectivity for vagal afferent over efferent targeting than traditional approaches. It can be used to address questions about the role of gut to brain signaling in physiological and pathophysiological conditions.
Subject(s)
Afferent Pathways/drug effects , Autonomic Denervation/methods , Gastrointestinal Tract/drug effects , Nerve Block/methods , Ribosome Inactivating Proteins, Type 1/administration & dosage , Vagus Nerve/drug effects , Afferent Pathways/physiology , Animals , Gastrointestinal Tract/physiology , Male , Neurotoxins/administration & dosage , Neurotoxins/pharmacology , Rats , Rats, Wistar , Saporins , Treatment Outcome , Vagus Nerve/physiologyABSTRACT
Therapeutically potent macromolecular drugs have shown great promise for overcoming the limitations of small-molecule anti-cancer drugs. But tumor cell-selective intracellular delivery of the macromolecules remains a major hurdle for their successful clinical application. To overcome this challenge, we engineered a novel genetic fusion protein (F3-Gel) that composed of F3 peptide, a tumor-homing peptide, and gelonin, a plant-derived ribosome-inactivating protein (RIP), and then evaluated its anti-cancer activity in vitro and in vivo. The F3-Gel-encoding gene was synthesized by genetic recombination, and F3-Gel was successfully expressed in E coli. The anti-cancer activity of the produced F3-Gel was evaluated by various in vitro assays, which revealed that F3-Gel maintained equipotent protein synthesis inhibition activity (IC50=11 pmol/L) as unmodified gelonin (IC50=10 pmol/L). Furthermore, F3-Gel displayed enhanced cellular uptake into cancer cells (U87 MG, HeLa, LnCaP and 9L) than noncancerous cells (293 HEK and SVGp12). Compared with gelonin, F3-Gel exerted significantly higher cytotoxicity against these cancer cells. F3-Gel displayed significantly greater inhibition of protein translation in U87 MG cells: F3-Gel (0.5 µmol/L) was able to reduce the protein level to less than 50%, while gelonin (1 µmol/L) did not affect the intracellular protein level. In a U87 MG xenograft tumor-bearing mouse model, F3-Gel was accumulated in the tumor site at much higher levels and maintained for a prolonged time compared with gelonin. Administration of F3-Gel (0.5, 0.75 mol/kg, iv) caused 36% and 66%, respectively, inhibition of tumor growth in U87 MG xenograft mice, suggesting that it is a promising candidate drug for cancer treatment. Furthermore, this study demonstrates that fusion of F3 peptide to a potent macromolecule could provides an effective method for targeting tumors and eventually could improve their druggability.
Subject(s)
Antineoplastic Agents/pharmacology , Peptides/pharmacology , Ribosome Inactivating Proteins, Type 1/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Gels/administration & dosage , Gels/chemistry , Gels/pharmacology , Humans , Injections, Intravenous , Macromolecular Substances/chemistry , Macromolecular Substances/pharmacology , Male , Mice , Mice, Nude , Neoplasms, Experimental/diagnosis , Neoplasms, Experimental/drug therapy , Peptides/administration & dosage , Peptides/chemistry , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Ribosome Inactivating Proteins, Type 1/administration & dosage , Ribosome Inactivating Proteins, Type 1/chemistry , Structure-Activity RelationshipABSTRACT
BACKGROUND: The possibility of using deep brain stimulation (DBS) for memory enhancement has recently been reported, but the precise underlying mechanisms of its effects remain unknown. Our previous study suggested that spatial memory improvement by medial septum (MS)-DBS may be associated with cholinergic regulation and neurogenesis. However, the affected stage of memory could not be distinguished because the stimulation was delivered during the execution of all memory processes. Therefore, this study was performed to determine the stage of memory affected by MS-DBS. Rats were administered 192 IgG-saporin to lesion cholinergic neurons. Stimulation was delivered at different times in different groups of rats: 5 days before the Morris water maze test (pre-stimulation), 5 days during the training phase of the Morris water maze test (training-stimulation), and 2 h before the Morris water maze probe test (probe-stimulation). A fourth group of rats was lesioned but received no stimulation. These four groups were compared with a normal (control) group. RESULTS: The most effective memory restoration occurred in the pre-stimulation group. Moreover, the pre-stimulation group exhibited better recall of the platform position than the other stimulation groups. An increase in the level of brain derived neurotrophic factor (BDNF) was observed in the pre-stimulation group; this increase was maintained for 1 week. However, acetylcholinesterase activity in the pre-stimulation group was not significantly different from the lesion group. CONCLUSION: Memory impairment due to cholinergic denervation can be improved by DBS. The improvement is significantly correlated with the up-regulation of BDNF expression and neurogenesis. Based on the results of this study, the use of MS-DBS during the early stage of disease may restore spatial memory impairment.
Subject(s)
Deep Brain Stimulation/methods , Maze Learning/physiology , Septal Nuclei/physiology , Spatial Memory/physiology , Animals , Antibodies, Monoclonal/administration & dosage , Brain-Derived Neurotrophic Factor/metabolism , Choline O-Acetyltransferase/metabolism , Cholinergic Agents/administration & dosage , Cholinergic Neurons/drug effects , Cholinergic Neurons/metabolism , Glutamate Decarboxylase/metabolism , Male , Neurogenesis , Rats , Rats, Sprague-Dawley , Ribosome Inactivating Proteins, Type 1/administration & dosage , Saporins , Septal Nuclei/drug effects , Septal Nuclei/metabolismABSTRACT
Oxytocin (OXT)-containing neurosecretory cells in the parvocellular divisions of the paraventricular nucleus (PVN), which project to the medulla and spinal cord, are involved in various physiological functions, such as sensory modulation and autonomic processes. In the present study, we examined OXT expression in the hypothalamo-spinal pathway, as well as the hypothalamo-neurohypophysial system, which includes the magnocellular neurosecretory cells in the PVN and the supraoptic nucleus (SON), after s.c. injection of saline or formalin into the hindpaws of transgenic rats that express the OXT and monomeric red fluorescent protein 1 (mRFP1) fusion gene. (i) The numbers of OXT-mRFP1 neurones that expressed Fos-like immunoreactivity (-IR) and OXT-mRFP1 intensity were increased significantly in the magnocellular/parvocellular PVN and SON after s.c. injection of formalin. (ii) OXT-mRFP1 neurones in the anterior parvocellular PVN, which may project to the dorsal horn of the spinal cord, were activated by s.c. injection of formalin, as indicated by a significant increases of Fos-IR and mRFP1 intensity intensity. (iii) Formalin injection caused a significant transient increase in plasma OXT. (iv) OXT, mRFP1 and corticotrophin-releasing hormone mRNAs in the PVN were significantly increased after s.c. injection of formalin. (v) An intrathecal injection of OXT-saporin induced hypersensitivity in conscious rats. Taken together, these results suggest that the hypothalamo-neurohypophysial/-spinal OXTergic pathways may be involved in acute nociceptive responses in rats.
Subject(s)
Hyperalgesia/chemically induced , Hyperalgesia/physiopathology , Hypothalamus/metabolism , Oxytocin/physiology , Pituitary Gland, Posterior/metabolism , Animals , Corticotropin-Releasing Hormone/biosynthesis , Formaldehyde , Injections, Spinal , Luminescent Proteins/genetics , Male , Neurons/metabolism , Oxytocin/administration & dosage , Oxytocin/analogs & derivatives , Oxytocin/biosynthesis , Oxytocin/blood , Oxytocin/pharmacology , Pain Measurement , Paraventricular Hypothalamic Nucleus/metabolism , Paraventricular Hypothalamic Nucleus/physiology , Proto-Oncogene Proteins c-fos/metabolism , Rats , Rats, Transgenic , Ribosome Inactivating Proteins, Type 1/administration & dosage , Ribosome Inactivating Proteins, Type 1/pharmacology , Saporins , Supraoptic Nucleus/metabolism , Supraoptic Nucleus/physiology , Red Fluorescent ProteinABSTRACT
Alzheimer's disease (AD) is a disease of complex etiology, involving multiple risk factors. When these risk factors are presented concomitantly, cognition and brain pathology are more severely compromised than if those risk factors were presented in isolation. Reduced cholinergic tone and elevated amyloid-beta (Aß) load are pathological hallmarks of AD. The present study sought to investigate brain pathology and alterations in learning and memory when these two factors were presented together in rats. Rats received either sham surgeries, cholinergic depletions of the medial septum, intracerebroventricular Aß25-35 injections, or both cholinergic depletion and Aß25-35 injections (Aß+ACh group). The Aß+ACh rats were unimpaired in a striatal dependent visual discrimination task, but had impaired acquisition in the standard version of the Morris water task. However, these rats displayed normal Morris water task retention and no impairment in acquisition of a novel platform location during a single massed training session. Aß+ACh rats did not have exacerbated brain pathology as indicated by activated astroglia, activated microglia, or accumulation of Aß. These data suggest that cholinergic depletions and Aß injections elicit subtle cognitive deficits when behavioural testing is conducted shortly after the presentation of these factors. These factors might have altered hippocampal synaptic plasticity and thus resemble early AD pathology.
Subject(s)
Acetylcholine/physiology , Alzheimer Disease/physiopathology , Alzheimer Disease/psychology , Amyloid beta-Peptides/metabolism , Learning , Memory , Peptide Fragments/metabolism , Alzheimer Disease/chemically induced , Alzheimer Disease/metabolism , Amyloid beta-Peptides/administration & dosage , Animals , Antibodies, Monoclonal/administration & dosage , Astrocytes/drug effects , Astrocytes/metabolism , Brain/drug effects , Brain/metabolism , Choline O-Acetyltransferase/metabolism , Cholinergic Agents/administration & dosage , Cholinergic Neurons/drug effects , Cholinergic Neurons/metabolism , Cognition/drug effects , Discrimination, Psychological/drug effects , Disease Models, Animal , Learning/drug effects , Male , Maze Learning/drug effects , Memory/drug effects , Microglia/drug effects , Microglia/metabolism , Peptide Fragments/administration & dosage , Rats , Rats, Long-Evans , Ribosome Inactivating Proteins, Type 1/administration & dosage , Saporins , Septal Nuclei/drug effects , Septal Nuclei/metabolismSubject(s)
Autonomic Nervous System/surgery , Catheter Ablation/methods , Death, Sudden, Cardiac/prevention & control , Heart/innervation , Neurons/pathology , Sympathectomy/methods , Anti-Arrhythmia Agents/adverse effects , Anti-Arrhythmia Agents/therapeutic use , Arrhythmias, Cardiac/drug therapy , Arrhythmias, Cardiac/physiopathology , Arrhythmias, Cardiac/prevention & control , Autonomic Nervous System/anatomy & histology , Catheter Ablation/adverse effects , Cholera Toxin/administration & dosage , Death, Sudden, Cardiac/pathology , Heart/physiopathology , Humans , Immunotoxins/administration & dosage , Incidence , Neurons/drug effects , Ribosome Inactivating Proteins, Type 1/administration & dosage , Saporins , Stellate Ganglion/drug effects , Stellate Ganglion/pathology , Sympathectomy/adverse effects , Thoracic Surgical Procedures/adverse effects , Thoracic Surgical Procedures/methodsABSTRACT
Dementia of the Alzheimer's type (DAT) is a neurodegenerative disorder marked by loss of hippocampal cholinergic tone and significant memory impairments, specifically for memories acquired prior to disease onset. The nature of this relationship, however, remains debated. The current study used the string pulling task to evaluate the temporal effects of odor discrimination learning in animals with selective cholinergic lesions to determine the role of the septohippocampal cholinergic system in mnemonic function. Rats with 192-IgG-Saporin lesions to the medial septum had a higher number of correct responses in the reversal training when compared to sham rats, suggesting an inability to retrieve the previously learned discrimination; however, no temporal gradient was observed. Furthermore, there were no group differences when learning a novel odor discrimination, demonstrating the ability for all rats to form new memories. These results establish a role for the cholinergic medial septum projections in long-term memory retrieval. The current study provides a behavioral assessment technique to investigate factors that influence mnemonic deficits associated with rodent models of DAT.
Subject(s)
Amnesia, Retrograde/physiopathology , Discrimination Learning/physiology , Hippocampus/drug effects , Memory, Long-Term/physiology , Odorants , Amnesia, Retrograde/chemically induced , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/pharmacology , Cholinergic Agents/administration & dosage , Cholinergic Agents/pharmacology , Female , Memory Disorders/chemically induced , Memory Disorders/pathology , Rats , Rats, Long-Evans , Ribosome Inactivating Proteins, Type 1/administration & dosage , Ribosome Inactivating Proteins, Type 1/pharmacology , SaporinsABSTRACT
BACKGROUND: Non-peptidergic nociceptive neurons are a sub-population of small diameter primary sensory neurons that comprise approximately 50 % of the C fiber population. Together with the peptidergic sub-population, they transmit nociceptive information from the periphery to the superficial dorsal horn of the spinal cord. Despite the numerous studies investigating the role of the non-peptidergic primary afferents, their role in normal nociception and in pain remains poorly understood. Our lab has previously demonstrated that, in rat models of neuropathic and inflammatory pain, there is a de novo expression of substance P receptors (NK-1r) by lamina I pyramidal projection neurons, a neuronal population that normally does not express these receptors. RESULTS: In this study, we used a ribosomal toxin, saporin, conjugated to the lectin IB4 to selectively ablate the non-peptidergic nociceptive C fibers, to investigate if the loss of these fibers was enough to induce a change in NK-1r expression by lamina I projection neurons. IB4-saporin treatment led to the permanent ablation of the IB4-positive afferents but also to a small non-significant reduction in CGRP-positive afferents. An overall increase in immunoreactivity for the NK-1r was observed in lamina I projection neurons, however, the lack of non-peptidergic afferents did not increase the number of lamina I pyramidal projection neurons immunoreactive for the receptor. CONCLUSIONS: Our results demonstrate that the deletion of the non-peptidergic afferents, at the L4-L5 spinal levels, is not sufficient to trigger the de novo expression of NK-1r by projection pyramidal neurons but increases the expression of NK-1r in fusiform and multipolar projection neurons. Furthermore, our data suggest that a neuropathic component is essential to trigger the expression of NK-1r by pyramidal neurons.
Subject(s)
Nerve Fibers, Unmyelinated/metabolism , Peptides/metabolism , Posterior Horn Cells/metabolism , Animals , Behavior, Animal/drug effects , Injections , Lectins/administration & dosage , Lectins/pharmacology , Male , Microscopy, Confocal , Nerve Fibers, Unmyelinated/drug effects , Posterior Horn Cells/drug effects , Rats, Sprague-Dawley , Ribosome Inactivating Proteins, Type 1/administration & dosage , Ribosome Inactivating Proteins, Type 1/pharmacology , SaporinsABSTRACT
Both lateral hypothalamic orexinergic neurons and hindbrain catecholaminergic neurons contribute to control of feeding behavior. Orexin fibers and terminals are present in close proximity to hindbrain catecholaminergic neurons, and fourth ventricular (4V) orexin injections that increase food intake also increase c-Fos expression in hindbrain catecholamine neurons, suggesting that orexin neurons may stimulate feeding by activating catecholamine neurons. Here we examine that hypothesis in more detail. We found that 4V injection of orexin-A (0.5 nmol/rat) produced widespread activation of c-Fos in hindbrain catecholamine cell groups. In the A1 and C1 cell groups in the ventrolateral medulla, where most c-Fos-positive neurons were also dopamine ß hydroxylase (DBH) positive, direct injections of a lower dose (67 pmol/200 nl) of orexin-A also increased food intake in intact rats. Then, with the use of the retrogradely transported immunotoxin, anti-DBH conjugated to saporin (DSAP), which targets and destroys DBH-expressing catecholamine neurons, we examined the hypothesis that catecholamine neurons are required for orexin-induced feeding. Rats given paraventricular hypothalamic injections of DSAP, or unconjugated saporin (SAP) as control, were implanted with 4V or lateral ventricular (LV) cannulas and tested for feeding in response to ventricular injection of orexin-A (0.5 nmol/rat). Both LV and 4V orexin-A stimulated feeding in SAP controls, but DSAP abolished these responses. These results reveal for the first time that catecholamine neurons are required for feeding induced by injection of orexin-A into either LV or 4V.
Subject(s)
Catecholamines/metabolism , Cerebral Ventricles/drug effects , Eating/drug effects , Feeding Behavior/drug effects , Intracellular Signaling Peptides and Proteins/administration & dosage , Nerve Fibers/drug effects , Neuropeptides/administration & dosage , Rhombencephalon/drug effects , Animals , Cerebral Ventricles/cytology , Cerebral Ventricles/immunology , Cerebral Ventricles/metabolism , Dopamine beta-Hydroxylase/immunology , Dopamine beta-Hydroxylase/metabolism , Immunotoxins/administration & dosage , Injections, Intraventricular , Male , Nerve Fibers/immunology , Nerve Fibers/metabolism , Orexins , Proto-Oncogene Proteins c-fos/metabolism , Rats, Sprague-Dawley , Rhombencephalon/cytology , Rhombencephalon/immunology , Rhombencephalon/metabolism , Ribosome Inactivating Proteins, Type 1/administration & dosage , SaporinsABSTRACT
The use of toxins for cancer therapy has great promise. Gelonin, a potent plant toxin, causes cell death by inactivating the 60S ribosomal subunit. Recently, we developed a novel gene delivery system using biodegradable cationic heparin-polyethyleneimine (HPEI) nanogels. In the current study, the antitumor activity of a recombinant plasmid expressing gelonin (pGelonin) on human ovarian cancer was assessed. The application of HPEI nanogels, was also evaluated. Gelonin-cDNA was cloned into the pVAX1 plasmid vector and transfected into SKOV3 human ovarian cancer cells using biodegradable cationic HPEI nanogels. The expression of gelonin in vitro and in vivo was confirmed using RT-PCR and western blot analysis. Cell viability and apoptosis were examined using an MTT assay and flow cytometric analysis. For the in vivo study, an SKOV3 intraperitoneal ovarian carcinomatosis model was established, and nude mice were randomly assigned into four groups receiving i.p. administration of pGelonin/HPEI complexes, pVAX/HPEI complexes, HPEI alone and 5% glucose solution. The tumor weight was monitored, and a TUNEL assay and Ki-67 immunohistochemistry were performed to evaluate apoptosis and cell proliferation in the tumor tissue sections, respectively. Gelonin was efficiently expressed in SKOV3 cancer cells in vitro and in vivo using pGelonin incorporated with HPEI nanogels. The pGelonin/HPEI complexes inhibited cell viability and induced apoptosis in the cell culture. Treatment for intraperitoneal carcinomatosis with pGelonin/HPEI complexes reduced the tumor weight by ~58.55% compared to the control groups (P<0.05). The antitumor effect was accompanied by increased apoptosis and reduced cell proliferation (P<0.05). No significant side effects were observed with i.p. administration of the pGelonin/HPEI complexes. Our data indicate that HPEI nanogel-delivered pGelonin may have promising applications against human ovarian cancer.
Subject(s)
Genetic Therapy/methods , Heparin/chemistry , Ovarian Neoplasms/therapy , Polyethylene Glycols/administration & dosage , Polyethyleneimine/administration & dosage , Ribosome Inactivating Proteins, Type 1/pharmacology , Animals , Apoptosis/genetics , Biocompatible Materials , Cations , Cell Line , Female , Gene Transfer Techniques , Heparin/administration & dosage , Humans , Mice, Inbred BALB C , Mice, Nude , Nanogels , Ovarian Neoplasms/pathology , Polyethylene Glycols/chemistry , Polyethylene Glycols/toxicity , Polyethyleneimine/chemistry , Polyethyleneimine/toxicity , Ribosome Inactivating Proteins, Type 1/administration & dosage , Ribosome Inactivating Proteins, Type 1/genetics , Xenograft Model Antitumor AssaysABSTRACT
The aim of this study was to investigate the role of the medial septal (MS) GABAergic cells in hippocampal dependent spatial learning using the immunotoxin GAT1-SAP to produce selective lesions of GABAergic MS neurons. In current study rats were trained in a visible platform version of the Morris water maze in which either a place or cue strategy could be used to escape successfully. Immunohistochemical studies showed that intraseptal injection of GAT1-SAP extensively damaged GABAergic MS neurons and spared most cholinergic neurons. The rats' responses on the competition test were classified as either cue or place, based on the swim path for those trials. An overview of the data from both competition trials for each group show that the control rats in 14 trials out of 16 competition test trial used place strategy, while MS-lesioned ones used this strategy in 2 trials only. Decreased place-bias in MS-lesioned rats compared to the control rats was significant (P<0.01). The data obtained in the control and GAT1-SAP lesioned animals in the present study, demonstrate that lesioned rats were impaired in hidden platform trials during training, and displayed a pronounced cue-bias in competition tests. Therefore, above data suggest involvement of the MS GABAergic neurons in organization of the spatial map-driven behavior and this structure, along with the hippocampus, should be viewed as a constituent of the functional system responsible for the cognitive types of spatial memory.
Subject(s)
GABAergic Neurons/pathology , Ribosome Inactivating Proteins, Type 1/administration & dosage , Septal Nuclei/physiopathology , Spatial Learning/drug effects , Animals , GABAergic Neurons/drug effects , Humans , Immunotoxins/administration & dosage , Maze Learning/drug effects , Rats , Saporins , Septal Nuclei/drug effects , gamma-Aminobutyric Acid/metabolismABSTRACT
The cancer stem cell (CSC) marker CD133 is an attractive target to improve antitumor therapy. We have used photochemical internalization (PCI) for the endosomal escape of the novel CD133-targeting immunotoxin AC133-saporin (PCIAC133-saporin). PCI employs an endocytic vesicle-localizing photosensitizer, which generates reactive oxygen species upon light-activation causing a rupture of the vesicle membranes and endosomal escape of entrapped drugs. Here we show that AC133-saporin co-localizes with the PCI-photosensitizer TPCS2a, which upon light exposure induces cytosolic release of AC133-saporin. PCI of picomolar levels of AC133-saporin in colorectal adenocarcinoma WiDr cells blocked cell proliferation and induced 100% inhibition of cell viability and colony forming ability at the highest light doses, whereas no cytotoxicity was obtained in the absence of light. Efficient PCI-based CD133-targeting was in addition demonstrated in the stem-cell-like, triple negative breast cancer cell line MDA-MB-231 and in the aggressive malignant melanoma cell line FEMX-1, whereas no enhanced targeting was obtained in the CD133-negative breast cancer cell line MCF-7. PCIAC133-saporin induced mainly necrosis and a minimal apoptotic response based on assessing cleavage of caspase-3 and PARP, and the TUNEL assay. PCIAC133-saporin resulted in S phase arrest and reduced LC3-II conversion compared to control treatments. Notably, co-treatment with Bafilomycin A1 and PCIAC133-saporin blocked LC3-II conversion, indicating a termination of the autophagic flux in WiDr cells. For the first time, we demonstrate laser-controlled targeting of CD133 in vivo. After only one systemic injection of AC133-saporin and TPCS2a, a strong anti-tumor response was observed after PCIAC133-saporin. The present PCI-based endosomal escape technology represents a minimally invasive strategy for spatio-temporal, light-controlled targeting of CD133+ cells in localized primary tumors or metastasis.
Subject(s)
Adenocarcinoma/drug therapy , Colorectal Neoplasms/drug therapy , Immunotoxins/administration & dosage , Photosensitizing Agents/administration & dosage , Ribosome Inactivating Proteins, Type 1/administration & dosage , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Cell Line, Tumor , Colon/drug effects , Colon/metabolism , Colon/pathology , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Drug Delivery Systems , Endosomes/drug effects , Endosomes/metabolism , Humans , Immunotoxins/metabolism , Immunotoxins/pharmacology , Light , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Photochemotherapy , Photosensitizing Agents/metabolism , Photosensitizing Agents/pharmacology , Rectum/drug effects , Rectum/metabolism , Rectum/pathology , Ribosome Inactivating Proteins, Type 1/metabolism , Ribosome Inactivating Proteins, Type 1/pharmacology , SaporinsABSTRACT
In the present study electrolytic and the immunotoxins (192 IgG saporin and GAT1-SAP) lesions of medial septal area (MS) were used to investigate the importance of cholinergic and GABAergic MS neurons in spatial working memory using spatial alternation task. In our experiments electrolytic lesions destroyed on average 69% of the intact MS. Examination of the AChE stained sections showed that after injections of 192 IgG saporin into the MS, animals exhibited significantly less AChE staining in MS as compared to sections obtained from control animals. Intraseptal GAT1-SAP preferentially reduced GABAergic neurons as compared to cholinergic neurons in the MS. The results of present study indicate that spatial short-term memory is affected only by electrolytic but not 192 IgG saporin or GAT1-SAP lesions. The behavioral testing showed that 192 IgG saporin treated rats, relative to control rats, had a significantly lower level in the number of arms entered during the testing session. However, the groups did not differ in the level of alternation behavior. GAT1-SAP lesioned rats showed that the percent alternation scores and the number of arms that the rat entered in the maze were not significantly different from control rats. These findings indicate that deficits observed after septal electrolytic lesions cannot be accounted solely to the loss of cholinergic or GABAergic septohippocampal projections. To determine more definitively whether septohippocampal projection neurons are required for the spatial short-term memory it would be ideal to produce in future combined lesions of the cholinergic and GABA-ergic septohippocampal projection neurons using 192 IgG-saporin and GAT1-SAP.
Subject(s)
Memory, Short-Term/physiology , Neurons/physiology , Septum of Brain/physiology , Acetylcholine/metabolism , Animals , Antibodies, Monoclonal/administration & dosage , Hippocampus/drug effects , Hippocampus/physiology , Humans , Immunotoxins/administration & dosage , Male , Maze Learning/drug effects , Memory, Short-Term/drug effects , Neurons/drug effects , Rats , Ribosome Inactivating Proteins, Type 1/administration & dosage , Saporins , Septum of Brain/drug effects , gamma-Aminobutyric Acid/metabolismABSTRACT
Traditionally, any drug intended for combating the tumor would distribute profoundly to other organs and tissues as lack of targeting specificity, thus resulting in limited therapeutic effects toward the tumor but severe drug-induced toxic side effects. To prevail over this obstacle of drug-induced systemic toxicity, a novel approach termed "ATTEMPTS" (antibody targeted triggered electrically modified prodrug type strategy) was designed, which directly introduces both of the targeting and prodrug features onto the protein drugs. The ATTEMPTS system is composed of the antibody targeting component consisting of antibodies linked with heparin, and the cell penetrating peptide (CPP) modified drug component. The two components mentioned above self-assembled into a tight complex via the charge to charge interaction between the anionic heparin and cationic CPP. Once accumulated at the targeting site, the CPP modified drug is released from the blockage by a second triggering agent, while remaining inactive in the circulation during tumor targeting thus aborting its effect on normal tissues. We utilized the heparin-induced inhibition on the cell-penetrating activity of CPP to create the prodrug feature, and subsequently the protamine-induced reversal of heparin inhibition to resume cell transduction of the protein drug via the CPP function. Our approach is the first known system to overcome this selectivity issue, enabling CPP-mediated cellular drug delivery to be practically applicable clinically. In this review, we thoroughly discussed the historical and novel progress of the "ATTEMPTS" system.
Subject(s)
Antibodies/metabolism , Antineoplastic Agents/administration & dosage , Cell-Penetrating Peptides/metabolism , Drug Delivery Systems , Heparin/metabolism , Immunoconjugates/administration & dosage , Peptide Fragments/metabolism , Prodrugs/administration & dosage , Ribosome Inactivating Proteins, Type 1/administration & dosage , tat Gene Products, Human Immunodeficiency Virus/metabolism , Animals , Antibodies/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/toxicity , Cell-Penetrating Peptides/chemistry , Chemistry, Pharmaceutical , Drug Carriers , Drug Delivery Systems/history , Drug Delivery Systems/trends , Heparin/chemistry , History, 21st Century , Humans , Immunoconjugates/chemistry , Immunoconjugates/metabolism , Immunoconjugates/toxicity , Peptide Fragments/chemistry , Prodrugs/chemistry , Prodrugs/metabolism , Prodrugs/toxicity , Ribosome Inactivating Proteins, Type 1/chemistry , Ribosome Inactivating Proteins, Type 1/metabolism , Ribosome Inactivating Proteins, Type 1/toxicity , Technology, Pharmaceutical/methods , Xenograft Model Antitumor Assays , tat Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
CONTEXT: Saponinum album (SA) is a complex mixture of triterpenoid saponins previously shown to augment the cytotoxicity of the type I ribosome-inactivating protein saporin and an EGF-saporin target toxin that could potentially be used to improve the therapeutic window of targeted toxins. OBJECTIVE: To investigate the augmentative property of SA on saporin and saporin-based immunotoxins (IT) directed against five different cell surface target molecules on human leukemia and lymphoma cells. MATERIALS AND METHODS: After determining the optimum dose of SA for each cell line, the extent of SA-mediated augmentation was established for saporin and five saporin-based ITs using XTT and an annexin V apoptosis assay. Immunospecificity was investigated using three different blocking assays. Dose-scheduling was also investigated using the XTT assay. RESULTS: Uncorrected SA-mediated augmentation ranged at best from 31.5 million-fold to, at worse, 174-fold. However, when the calculated fold-increases were adjusted for the non-immunospecific effects of SA on an off-target IT, the true augmentative effects of SA were found to be largely non-immunospecific. Antibody blocking studies demonstrated that the augmentative effect of SA was only partially immunospecific. Separate exposure of target cells to IT and SA at different times demonstrated that immunospecific augmentation of IT by SA could be achieved but only if cells were exposed to IT first and SA second. CONCLUSIONS: SA significantly, although variably, augments the cytotoxicity of saporin and saporin-based immunotoxins. Concomitant exposure to both IT and SA can result in non-immunospecific cytotoxicity that can be overcome by temporally separating exposure to each.