ABSTRACT
BACKGROUND: Vascular Endothelial Growth Factor Receptors (VEGFR1 and VEGFR2) are tyrosine kinase receptors expressed on endothelial cells and tumor vessels and play an important role in angiogenesis. In this study, three repeats of VEGFR1 and VEGFR2 binding peptide (VGB3) were genetically fused to the truncated diphtheria toxin (TDT), and its in vitro activity was evaluated. METHODS: The recombinant construct (TDT-triVGB3) was expressed in bacteria cells and purified with nickel affinity chromatography. The binding capacity and affinity of TDT-triVGB3 were evaluated using the enzyme-linked immunosorbent assay. The inhibitory activity of TDT-triVGB3 on viability, migration, and tube formation of human endothelial cells was evaluated using MTT, migration, and tube formation assays. RESULTS: TDT-triVGB3 selectively detected VEGFR1 and VEGFR2 with high affinity in an enzyme- linked immunosorbent assay and significantly inhibited viability, migration, and tube formation of human endothelial cells. CONCLUSION: The developed TDT-triVGB3 is potentially a novel agent for targeting VEGFR1/ VEGFR2 over-expressing cancer cells.
Subject(s)
Angiogenesis Inhibitors , Cell Movement , Diphtheria Toxin , Human Umbilical Vein Endothelial Cells , Vascular Endothelial Growth Factor Receptor-1 , Vascular Endothelial Growth Factor Receptor-2 , Humans , Vascular Endothelial Growth Factor Receptor-1/genetics , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism , Diphtheria Toxin/genetics , Diphtheria Toxin/pharmacology , Diphtheria Toxin/metabolism , Cell Movement/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/genetics , Angiogenesis Inhibitors/chemistry , Cell Survival/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/metabolism , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Neovascularization, Pathologic/drug therapy , Gene Expression , Endothelial Cells/metabolism , Endothelial Cells/drug effectsABSTRACT
BACKGROUND: Targeted cancer therapy can be considered as a new strategy to overcome the side effects of current cancer treatments. Neuropilin-1 (NRP-1) is a transmembrane glycoprotein that is expressed in endothelial cells and tumor vessels to stimulate angiogenesis progression. Targeted diphtheria toxin (DT)- based therapeutics are promising tools for cancer treatment. This study aimed to construct a novel NRP-1 binding peptide (as three repeats) (CRGDK) as a fusion to truncated DT (DTA) (DTA-triCRGDK) for targeted delivery of DT into NRP-1 expressing cells. METHODS: The concept of DTA-triCRGDK was designed, synthesized and cloned into the bacterial host. Expression of DTA-triCRGDK was induced by Isopropyl ß-D-1-thiogalactopyranoside (IPTG) and purification was performed using Ni-NTA chromatography. Biological activity of DTA-triCRGDK was evaluated using MTT, apoptosis, and wound healing assays. In addition, expression levels of apoptotic Bax, Bcl2, and Casp3 genes were determined by Real-time PCR. RESULTS: Cytotoxicity analysis showed the IC50 values of DTA-triCRGDK for A549 and MRC5 were 0.43 nM and 4.12 nM after 24 h, respectively. Bcl2 expression levels decreased 0.4 and 0.72 fold in A549 and MRC5, respectively. However, Bax and Casp3 expression level increased by 6.75 and 8.19 in A549 and 2.51 and 3.6 in MRC5 cells. CONCLUSION: Taken together, DTA-triCRGDK is a promising tool for targeted therapy of NRP-1 overexpressing cancer cells.
Subject(s)
Apoptosis , Carcinoma, Non-Small-Cell Lung , Diphtheria Toxin , Lung Neoplasms , Neuropilin-1 , Humans , Neuropilin-1/metabolism , Neuropilin-1/genetics , Apoptosis/drug effects , Diphtheria Toxin/pharmacology , Diphtheria Toxin/chemistry , Diphtheria Toxin/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/isolation & purification , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Dose-Response Relationship, Drug , A549 Cells , Peptides/pharmacology , Peptides/chemistryABSTRACT
The epidermal growth factor receptor (EGFR) remains one of the best molecules for developing targeted therapy for multiple human malignancies, including head and neck squamous cell carcinoma (HNSCC). Small molecule inhibitors or antibodies targeting EGFR have been extensively developed in recent decades. Immunotoxin (IT)based therapy, which combines cell surface binding ligands or antibodies with a peptide toxin, represents another cancer treatment option. A total of 3 diphtheria toxin (DT)based fusion toxins that target human EGFRmonovalent EGFR IT (monoEGFIT), bivalent EGFR IT (biEGFIT), and a bispecific IT targeting both EGFR and interleukin2 receptor (bisEGF/IL2IT) were recently generated by the authors. Improved efficacy and reduced toxicity of biEGFIT compared with monoEGFIT in immunocompromised HNSCC mouse models was reported. In the present study, bisEGF/IL2IT were generated using a unique DTresistant yeast expression system and evaluated the in vitro and in vivo efficacy and toxicity of the 3 EGFITs in immunocompetent mice. The results demonstrated that while the three EGFITs had different efficacies in vitro and in vivo against HNSCC, biEGFIT and bisEGF/IL2IT had significantly improved in vivo efficacy and remarkably less offtarget toxicity compared with monoEGFIT. In addition, bisEGF/IL2IT was superior to biEGFIT in reducing tumor size and prolonging survival in the metastatic model. These data suggested that targeting either the tumor immune microenvironment or enhancing the binding affinity could improve the efficacy of ITbased therapy. BiEGFIT and bisEGF/IL2IT represent improved candidates for ITbased therapy for future clinical development.
Subject(s)
Head and Neck Neoplasms , Interleukin-2 , Humans , Animals , Mice , Squamous Cell Carcinoma of Head and Neck/drug therapy , Cell Line, Tumor , ErbB Receptors/metabolism , Head and Neck Neoplasms/drug therapy , Diphtheria Toxin/pharmacology , Tumor MicroenvironmentABSTRACT
Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in a variety of cancers such as colon, stomach, pancreas, and prostate adenocarcinomas. Inhibition of EpCAM is considered as a potential target for cancer therapy. In current study, anti-EpCAM immunotoxin (α-EpCAM IT) was developed using genetic fusion of α-EpCAM single domain antibody (nanobody) (α-EpCAM Nb) to truncated form of diphtheria toxin. The expression of recombinant α-EpCAM IT was induced by Isopropyl ß-d-1-thiogalactopyranoside (IPTG) and confirmed by SDS-PAGE and western blot. Recombinant α-EpCAM IT was purified from the inclusion bodies and refolded using urea gradient procedure. The cytotoxicity and apoptosis activity of α-EpCAM IT on EpCAM over-expressing (MCF7), low-expressing (HEK293), and no-expressing (HUVEC) cells were evaluated by 3-4,5-Dimethylthiazol-2-yl (MTT) assay and annexin V-FITC-PI assay as well. In addition, anti-tumor activity of α-EpCAM IT was evaluated on nude mice bearing MCF7 tumor cells. Results showed success expression and purification of α-EpCAM IT. The α-EpCAM IT showed time and dose-dependent anti-proliferative activity on MCF-7 cells. However, α-EpCAM IT did not show any anti-proliferative activity on HEK293 and HUVEC cells as well. In addition, the annexin V-FITC-PI assay results showed that α-EpCAM IT significantly increased apoptotic rate in MCF-7 cells with no effect on HEK293 and HUVEC as well. Moreover, α-EpCAM IT significantly reduced tumor size in vivo study. The achieved results indicate the potential of designing α-EpCAM IT as a novel therapeutic for cancer therapy.
Subject(s)
Immunotoxins , Single-Domain Antibodies , Male , Animals , Mice , Humans , Epithelial Cell Adhesion Molecule/genetics , Immunotoxins/genetics , Immunotoxins/pharmacology , Diphtheria Toxin/genetics , Diphtheria Toxin/pharmacology , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Mice, Nude , HEK293 Cells , Cell Line, TumorABSTRACT
Specific cell ablation by the diphtheria toxin (DT) system is widely used to analyze the in vivo function of target cells in mice. In this chapter, we describe the methods of depleting regulatory T cells (Tregs) systemically or selectively in the skin. Since it has been difficult to conclude the importance of tissue-residing Tregs with systemic Treg ablation, we sought to selectively deplete cutaneous Tregs to investigate their function in the skin without the depletion of Tregs in non-target organs. Here, we describe protocols for the depletion of Tregs by the DT system, and subsequent analysis of Tregs in the skin and skin-draining lymph node (dLN) by flow cytometry. This procedure of selective depletion of cutaneous Tregs can be applicable to other tissues and cells, to allow investigation of the role of tissue-resident cells in mice.
Subject(s)
Diphtheria Toxin , T-Lymphocytes, Regulatory , Animals , Diphtheria Toxin/pharmacology , Forkhead Transcription Factors , Immunotherapy , Lymphocyte Depletion/methods , Mice , Mice, Inbred C57BLABSTRACT
Adaptive immune cells with regulatory function reportedly mediate immune escape in a variety of tumors. Little is known regarding the relevance of the most prominent regulatory cell populations, namely Foxp3+ T regulatory cells (Tregs) and CD19+IL-10+ B regulatory cells (Bregs), for neuroblastoma (NB) survival. After establishing a novel immunocompetent syngeneic NB mouse model where orthotopic tumors can be generated after intrarenal injection of NB975A cells, we studied the importance of Tregs and Bregs in Foxp3-DTR mice whose Tregs can be depleted by diphtheria toxin (DT) application as well as in CD19-specific IL-10 deficient mice that lack IL-10+ Bregs (CD19cre+/- × IL-10fl/fl mice). We observed Foxp3 Treg cells in tumors from wild type mice. On the contrary, Bregs or B cells were scarce. Specific depletion of Tregs in Foxp3-DTR mice resulted in an 85% reduction of tumor volume and weight compared to DT-treated wild type mice and untreated Foxp3-DTR mice. In contrast, NB tumor growth was not affected in CD19-specific IL-10 deficient mice. Similarly, mice lacking mature B cells (µMT mice) and CD19 deficient mice (CD19cre mice) showed no change in growth pattern of NB tumors. In Treg-depleted mice, reduced tumor growth was associated with an increased concentration of IFN-gamma, TNF-alpha, IL-4, IL-6, and IL-10 in isolated splenocytes. In summary, transient ablation of Tregs but not absence of Bregs hindered the growth of NB, strongly suggesting the therapeutic potential of targeting Tregs for this aggressive childhood tumor.
Subject(s)
B-Lymphocytes, Regulatory , Neuroblastoma , Animals , Antigens, CD19 , B-Lymphocytes, Regulatory/metabolism , Diphtheria Toxin/metabolism , Diphtheria Toxin/pharmacology , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Interleukin-10/metabolism , Interleukin-4/metabolism , Interleukin-6/metabolism , Mice , Mice, Inbred C57BL , Neuroblastoma/metabolism , T-Lymphocytes, Regulatory , Tumor Necrosis Factor-alpha/metabolismABSTRACT
High rates of relapsed and refractory diffuse large B-cell lymphoma (DLBCL) patients and life-threatening side effects associated with immunochemotherapy make an urgent need to develop new therapies for DLBCL patients. Immunotoxins seem very potent anticancer therapies but their use is limited because of their high toxicity. Accordingly, the self-assembling polypeptidic nanoparticle, T22-DITOX-H6, incorporating the diphtheria toxin and targeted to CXCR4 receptor, which is overexpressed in DLBCL cells, could offer a new strategy to selectively eliminate CXCR4+ DLBCL cells without adverse effects. In these terms, our work demonstrated that T22-DITOX-H6 showed high specific cytotoxicity towards CXCR4+ DLBCL cells at the low nanomolar range, which was dependent on caspase-3 cleavage, PARP activation and an increase of cells in early/late apoptosis. Repeated nanoparticle administration induced antineoplastic effect, in vivo and ex vivo, in a disseminated immunocompromised mouse model generated by intravenous injection of human luminescent CXCR4+ DLBCL cells. Moreover, T22-DITOX-H6 inhibited tumor growth in a subcutaneous immunocompetent mouse model bearing mouse CXCR4+ lymphoma cells in the absence of alterations in the hemogram, liver or kidney injury markers or on-target or off-target organ histology. Thus, T22-DITOX-H6 demonstrates a selective cytotoxicity towards CXCR4+ DLBCL cells without the induction of toxicity in non-lymphoma infiltrated organs nor hematologic toxicity.
Subject(s)
Antineoplastic Agents , Lymphoma, Large B-Cell, Diffuse , Nanoparticles , Receptors, CXCR4 , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Diphtheria Toxin/pharmacology , Disease Models, Animal , Heterografts , Humans , Immunocompetence , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/immunology , Lymphoma, Large B-Cell, Diffuse/metabolism , Mice , Receptors, CXCR4/metabolismABSTRACT
BACKGROUND: Recent studies have established that CCR2 (C-C chemokine receptor type 2) marks proinflammatory subsets of monocytes, macrophages, and dendritic cells that contribute to adverse left ventricle (LV) remodeling and heart failure progression. Elucidation of the effector mechanisms that mediate adverse effects of CCR2+ monocytes, macrophages, and dendritic cells will yield important insights into therapeutic strategies to suppress myocardial inflammation. METHODS: We used mouse models of reperfused myocardial infarction, angiotensin II and phenylephrine infusion, and diphtheria toxin cardiomyocyte ablation to investigate CCL17 (C-C chemokine ligand 17). We used Ccl17 knockout mice, flow cytometry, RNA sequencing, biochemical assays, cell trafficking studies, and in vivo cell depletion to identify the cell types that generate CCL17, define signaling pathways that controlled its expression, delineate the functional importance of CCL17 in adverse LV remodeling and heart failure progression, and determine the mechanistic basis by which CCL17 exerts its effects. RESULTS: We demonstrated that CCL17 is expressed in CCR2+ macrophages and cluster of differentiation 11b+ conventional dendritic cells after myocardial infarction, angiotensin II and phenylephrine infusion, and diphtheria toxin cardiomyocyte ablation. We clarified the transcriptional signature of CCL17+ macrophages and dendritic cells and identified granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling as a key regulator of CCL17 expression through cooperative activation of STAT5 (signal transducer and activator of transcription 5) and canonical NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells) signaling. Ccl17 deletion resulted in reduced LV remodeling, decreased myocardial fibrosis and cardiomyocyte hypertrophy, and improved LV systolic function after myocardial infarction and angiotensin II and phenylephrine infusion. We observed increased abundance of regulatory T cells (Tregs) in the myocardium of injured Ccl17 knockout mice. CCL17 inhibited Treg recruitment through biased activation of CCR4. CCL17 activated Gq signaling and CCL22 (C-C chemokine ligand 22) activated both Gq and ARRB (ß-arrestin) signaling downstream of CCR4. CCL17 competitively inhibited CCL22 stimulated ARRB signaling and Treg migration. We provide evidence that Tregs mediated the protective effects of Ccl17 deletion on myocardial inflammation and adverse LV remodeling. CONCLUSIONS: These findings identify CCL17 as a proinflammatory mediator of CCR2+ macrophages and dendritic cells and suggest that inhibition of CCL17 may serve as an effective strategy to promote Treg recruitment and suppress myocardial inflammation.
Subject(s)
Heart Failure , Myocardial Infarction , Angiotensin II/pharmacology , Animals , Chemokine CCL17/metabolism , Chemokine CCL17/pharmacology , Diphtheria Toxin/metabolism , Diphtheria Toxin/pharmacology , Heart Failure/genetics , Heart Failure/metabolism , Humans , Inflammation/metabolism , Ligands , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenylephrine/metabolism , Phenylephrine/pharmacology , T-Lymphocytes, Regulatory/metabolism , Ventricular RemodelingABSTRACT
Vasopressin-synthesizing neurons are located in several brain regions, including the hypothalamic paraventricular nucleus (PVN), supraoptic nucleus (SON) and suprachiasmatic nucleus (SCN). Vasopressin has been shown to have various functions in the brain, including social recognition memory, stress responses, emotional behaviors and circadian rhythms. The precise physiological functions of vasopressin-synthesizing neurons in specific brain regions remain to be clarified. Conditional ablation of local vasopressin-synthesizing neurons may be a useful tool for investigation of the functions of vasopressin neurons in the regions. In the present study, we characterized a transgenic rat line that expresses a mutated human diphtheria toxin receptor under control of the vasopressin gene promoter. Under a condition of salt loading, which activates the vasopressin gene in the hypothalamic PVN and SON, transgenic rats were i.c.v. injected with diphtheria toxin. Intracerebroventricular administration of diphtheria toxin after salt loading depleted vasopressin-immunoreactive cells in the hypothalamic PVN and SON, but not in the SCN. The number of oxytocin-immunoreactive cells in the hypothalamus was not significantly changed. The rats that received i.c.v. diphtheria toxin after salt loading showed polydipsia and polyuria, which were rescued by peripheral administration of 1-deamino-8-d-arginine vasopressin via an osmotic mini-pump. Intrahypothalamic administration of diphtheria toxin in transgenic rats under a normal hydration condition reduced the number of vasopressin-immunoreactive neurons, but not the number of oxytocin-immunoreactive neurons. The transgenic rat model can be used for selective ablation of vasopressin-synthesizing neurons and may be useful for clarifying roles of vasopressin neurons at least in the hypothalamic PVN and SON in the rat.
Subject(s)
Gene Transfer Techniques , Genes, Transgenic, Suicide , Neurons/metabolism , Vasopressins/metabolism , Animals , Apoptosis/drug effects , Apoptosis/genetics , Diphtheria Toxin/pharmacology , Gene Deletion , Genes, Transgenic, Suicide/drug effects , Heparin-binding EGF-like Growth Factor/genetics , Heparin-binding EGF-like Growth Factor/metabolism , Male , Neurons/drug effects , Paraventricular Hypothalamic Nucleus/drug effects , Paraventricular Hypothalamic Nucleus/metabolism , Rats , Rats, Inbred Lew , Rats, Transgenic , Supraoptic Nucleus/drug effects , Supraoptic Nucleus/metabolism , Vasopressins/geneticsABSTRACT
Rodent models of Parkinson's disease are based on transgenic expression of mutant synuclein, deletion of PD genes, injections of MPTP or rotenone, or seeding of synuclein fibrils. The models show histopathologic features of PD such as Lewi bodies but mostly only subtle in vivo manifestations or systemic toxicity. The models only partly mimic a predominant loss of dopaminergic neurons in the substantia nigra. We therefore generated mice that express the transgenic diphtheria toxin receptor (DTR) specifically in DA neurons by crossing DAT-Cre mice with Rosa26 loxP-STOP-loxP DTR mice. After defining a well-tolerated DTx dose, DAT-DTR and DTR-flfl controls were subjected to non-toxic DTx treatment (5 × 100 pg/g) and subsequent histology and behavioral tests. DAT protein levels were reduced in the midbrain, and tyrosine hydroxylase-positive neurons were reduced in the substantia nigra, whereas the pan-neuronal marker NeuN was not affected. Despite the promising histologic results, there was no difference in motor function tests or open field behavior. These are tests in which double mutant Pink1-/-SNCAA53T Parkinson mice show behavioral abnormalities. Higher doses of DTx were toxic in both groups. The data suggest that DTx treatment in mice with Cre/loxP-driven DAT-DTR expression leads to partial ablation of DA-neurons but without PD-reminiscent behavioral correlates.
Subject(s)
Heparin-binding EGF-like Growth Factor/genetics , Parkinson Disease/physiopathology , Animals , Brain/pathology , Corpus Striatum/metabolism , Diphtheria Toxin/metabolism , Diphtheria Toxin/pharmacology , Disease Models, Animal , Dopamine/metabolism , Dopaminergic Neurons/pathology , Female , Heparin-binding EGF-like Growth Factor/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Parkinson Disease/metabolism , Parkinsonian Disorders/chemically induced , Parkinsonian Disorders/metabolism , Substantia Nigra/pathology , Tyrosine 3-Monooxygenase/metabolism , alpha-Synuclein/metabolismABSTRACT
Immunotoxins, as a class of antitumor agents, consist of tumor-selective ligands linked to highly toxic protein molecules. This type of modified antibody has been designed for the therapy of cancers and a few viral infections. In this study, we designed immunotoxin consisting of mouse programmed cell death protein-1 (PD1), which genetically fused to diphtheria toxin (DT) subunit A (DT386). DNA construct was cloned, expressed in a bacterial system, purified, and confirmed by western blotting. The immunotoxin potency in the treatment of tumorous C57BL/6 mice was evaluated. Immunotoxin was injected intratumoral to mice, and through eight injections, 67% of the tumor volume of the test group started shrinking dramatically. On the contrary, the tumor size of the control group, treated with phosphate-buffered saline, continued its growth. The successful targeting of solid tumor cells by PD1-DT immunotoxin demonstrates the potential therapeutic utility of these conjugates.
Subject(s)
Diphtheria Toxin/pharmacology , Immunotoxins/pharmacology , Neoplasms/therapy , Programmed Cell Death 1 Receptor/genetics , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/immunology , Cell Survival , Diphtheria Toxin/genetics , Diphtheria Toxin/immunology , Humans , Immunotoxins/genetics , Immunotoxins/immunology , Mice , Neoplasms/immunology , Programmed Cell Death 1 Receptor/immunologyABSTRACT
Lack or dysfunction of the lymphatics leads to secondary lymphedema formation that seriously reduces the function of the affected organs and results in degradation of quality of life. Currently, there is no definitive treatment option for lymphedema. Here, we utilized nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) encoding murine Vascular Endothelial Growth Factor C (VEGFC) to stimulate lymphatic growth and function and reduce experimental lymphedema in mouse models. We demonstrated that administration of a single low-dose of VEGFC mRNA-LNPs induced durable, organ-specific lymphatic growth and formation of a functional lymphatic network. Importantly, VEGFC mRNA-LNP treatment reversed experimental lymphedema by restoring lymphatic function without inducing any obvious adverse events. Collectively, we present a novel application of the nucleoside-modified mRNA-LNP platform, describe a model for identifying the organ-specific physiological and pathophysiological roles of the lymphatics, and propose an efficient and safe treatment option that may serve as a novel therapeutic tool to reduce lymphedema.
Subject(s)
Lymphangiogenesis/genetics , Lymphatic Vessels/pathology , Lymphedema/pathology , Nucleosides/metabolism , Vascular Endothelial Growth Factor C/genetics , Animals , Blood Vessels/pathology , Cell Proliferation/drug effects , Diphtheria Toxin/pharmacology , Disease Models, Animal , HEK293 Cells , Humans , Immunity/drug effects , Injections, Intradermal , Lipids/administration & dosage , Lipids/chemistry , Lymphatic Vessels/drug effects , Mice, Inbred C57BL , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Organ Specificity , Poly C/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tamoxifen/pharmacology , Vascular Endothelial Growth Factor C/administration & dosage , Vascular Endothelial Growth Factor C/metabolismABSTRACT
Immunotoxins are protein-based drugs consist of a target-specific binding domain and a cytotoxic domain to eliminate target cells. Such compounds are potentially therapeutic to combat diseases such as cancer. Generally, the B-subunit of Shiga toxin (STXB) receptor, globotriaosylceramide (Gb3), is expressed in high amounts on a number of human tumors cancer cells. In this study, we evaluated a new antitumor candidate called DT389-STXB chimeric protein, which genetically fused the DT to B-subunit of Shiga-like toxin (STXB). First a chimeric protein, encoding DT389-STXB was synthesized. The optimized chimeric protein expressed in E.coli BL21 (DE3) and confirmed by anti-His Western blot analysis. T47D, SKBR3, 4T1 and MCF7 cell lines were treated separately with purified DT389-STXB recombinant protein and functional activity of DT389-STXB was analyzed by the cell enzyme-linked immunosorbentassay (ELISA), MTT, ICC, Western blot and apoptosis tests. The results indicated that the recombinant DT389-STXB fusion protein with a molecular weight of 53 kDa was successfully expressed in E.coli BL21 (DE3) and the anti-His western-blot was used to confirm the presence of the protein. The DT389-STXB fusion protein attached to T47D, SKBR3 and 4T1 cell lines with the proper affinity and induced dose-dependent cytotoxicity against GB3-expressing cancer cells in vitro. Our results showed that DT389-STXB fusion protein may be a promising candidate for antitumor therapy agent against breast cancer; however, further studies are required to explore its efficacy in vivo for therapeutic applications.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Diphtheria Toxin/pharmacology , Shiga Toxins/pharmacology , Animals , Apoptosis/drug effects , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Dose-Response Relationship, Drug , Female , Humans , MCF-7 Cells , Mice , Recombinant Fusion Proteins/pharmacology , Trihexosylceramides/metabolismABSTRACT
Cutaneous mast cells mediate numerous skin inflammatory processes and have anatomical and functional associations with sensory afferent neurons. We reveal that epidermal nerve endings from a subset of sensory nonpeptidergic neurons expressing MrgprD are reduced by the absence of Langerhans cells. Loss of epidermal innervation or ablation of MrgprD-expressing neurons increased expression of a mast cell gene module, including the activating receptor, Mrgprb2, resulting in increased mast cell degranulation and cutaneous inflammation in multiple disease models. Agonism of MrgprD-expressing neurons reduced expression of module genes and suppressed mast cell responses. MrgprD-expressing neurons released glutamate which was increased by MrgprD agonism. Inhibiting glutamate release or glutamate receptor binding yielded hyperresponsive mast cells with a genomic state similar to that in mice lacking MrgprD-expressing neurons. These data demonstrate that MrgprD-expressing neurons suppress mast cell hyperresponsiveness and skin inflammation via glutamate release, thereby revealing an unexpected neuroimmune mechanism maintaining cutaneous immune homeostasis.
Subject(s)
Glutamic Acid/metabolism , Mast Cells/metabolism , Neurons/metabolism , Skin/metabolism , Animals , Cells, Cultured , Dermatitis/metabolism , Dermatitis/pathology , Diphtheria Toxin/pharmacology , Disease Models, Animal , Female , Integrin beta Chains/genetics , Integrin beta Chains/metabolism , Langerhans Cells/cytology , Langerhans Cells/drug effects , Langerhans Cells/metabolism , Mast Cells/cytology , Mast Cells/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/cytology , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/deficiency , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Skin/pathology , beta-Alanine/chemistry , beta-Alanine/metabolism , beta-Alanine/pharmacologyABSTRACT
Pneumonia-induced lung injury and acute respiratory distress syndrome can develop because of an inappropriate inflammatory response to acute infections, leading to a compromised alveolar barrier. Recent work suggests that hospitalized patients with allergies/asthma are less likely to die of pulmonary infections and that there is a correlation between survival from acute respiratory distress syndrome and higher eosinophil counts; thus, we hypothesized that eosinophils associated with a type 2 immune response may protect against pneumonia-induced acute lung injury. To test this hypothesis, mice were treated with the type 2-initiating cytokine IL-33 intratracheally 3 days before induction of pneumonia with airway administration of a lethal dose of Staphylococcus aureus. Interestingly, IL-33 pretreatment promoted survival by inhibiting acute lung injury: amount of BAL fluid proinflammatory cytokines and pulmonary edema were both reduced, with an associated increase in oxygen saturation. Pulmonary neutrophilia was also reduced, whereas eosinophilia was strongly increased. This eosinophilia was key to protection; eosinophil reduction eliminated both IL-33-mediated protection against mortality and inhibition of neutrophilia and pulmonary edema. Together, these data reveal a novel role for eosinophils in protection against lung injury and suggest that modulation of pulmonary type 2 immunity may represent a novel therapeutic strategy.
Subject(s)
Acute Lung Injury/immunology , Eosinophils/immunology , Interleukin-33/immunology , Pneumonia, Staphylococcal/immunology , Pulmonary Edema/immunology , Respiratory Distress Syndrome/immunology , Staphylococcus aureus/pathogenicity , Acute Lung Injury/etiology , Acute Lung Injury/microbiology , Acute Lung Injury/prevention & control , Animals , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Diphtheria Toxin/pharmacology , Disease Models, Animal , Eosinophils/drug effects , Female , Gene Expression , Humans , Interleukin-33/genetics , Interleukin-33/pharmacology , Interleukin-5/deficiency , Interleukin-5/genetics , Interleukin-5/immunology , Leukocyte Count , Leukocyte Reduction Procedures , Mice , Mice, Inbred C57BL , Mice, Knockout , Neutrophils/drug effects , Neutrophils/immunology , Pneumonia, Staphylococcal/complications , Pneumonia, Staphylococcal/microbiology , Pneumonia, Staphylococcal/mortality , Pulmonary Edema/complications , Pulmonary Edema/microbiology , Pulmonary Edema/mortality , Respiratory Distress Syndrome/etiology , Respiratory Distress Syndrome/microbiology , Respiratory Distress Syndrome/prevention & control , Staphylococcus aureus/immunology , Survival AnalysisABSTRACT
The Hippo signaling pathway is a key regulator of tissue development and regeneration. Activation of the Hippo pathway leads to nuclear translocation of the YAP1 transcriptional coactivator, resulting in changes in gene expression and cell cycle entry. Recent studies have demonstrated the nuclear translocation of YAP1 during the development of the sensory organs of the inner ear, but the possible role of YAP1 in sensory regeneration of the inner ear is unclear. The present study characterized the cellular localization of YAP1 in the utricles of mice and chicks, both under normal conditions and after HC injury. During neonatal development, YAP1 expression was observed in the cytoplasm of supporting cells, and was transiently expressed in the cytoplasm of some differentiating hair cells. We also observed temporary nuclear translocation of YAP1 in supporting cells of the mouse utricle after short periods in organotypic culture. However, little or no nuclear translocation of YAP1 was observed in the utricles of neonatal or mature mice after ototoxic injury. In contrast, substantial YAP1 nuclear translocation was observed in the chicken utricle after streptomycin treatment in vitro and in vivo. Together, these data suggest that differences in YAP1 signaling may partially account for the differing regenerative abilities of the avian vs. mammalian inner ear.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Saccule and Utricle/embryology , Saccule and Utricle/injuries , Animals , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Chickens , Diphtheria Toxin/pharmacology , Female , Gene Expression Regulation, Developmental/drug effects , Hair Cells, Auditory/drug effects , Hair Cells, Auditory/metabolism , Homeodomain Proteins/metabolism , Humans , Male , Mice, Inbred C57BL , Mice, Transgenic , Protein Transport/drug effects , Saccule and Utricle/metabolism , Saccule and Utricle/pathology , Transcription Factor Brn-3C/metabolismABSTRACT
AIMS/HYPOTHESIS: The endocrine pancreas comprises the islets of Langerhans, primarily consisting of beta cells, alpha cells and delta cells responsible for secretion of insulin, glucagon and somatostatin, respectively. A certain level of intra-islet communication is thought to exist, where the individual hormones may reach the other islet cells and regulate their secretion. Glucagon has been demonstrated to importantly regulate insulin secretion, while somatostatin powerfully inhibits both insulin and glucagon secretion. In this study we investigated how secretion of somatostatin is regulated by paracrine signalling from glucagon and insulin. METHODS: Somatostatin secretion was measured from perfused mouse pancreases isolated from wild-type as well as diphtheria toxin-induced alpha cell knockdown, and global glucagon receptor knockout (Gcgr-/-) mice. We studied the effects of varying glucose concentrations together with infusions of arginine, glucagon, insulin and somatostatin, as well as infusions of antagonists of insulin, somatostatin and glucagon-like peptide 1 (GLP-1) receptors. RESULTS: A tonic inhibitory role of somatostatin was demonstrated with infusion of somatostatin receptor antagonists, which significantly increased glucagon secretion at low and high glucose, whereas insulin secretion was only increased at high glucose levels. Infusion of glucagon dose-dependently increased somatostatin secretion approximately twofold in control mice. Exogenous glucagon had no effect on somatostatin secretion in Gcgr-/- mice, and a reduced effect when combined with the GLP-1 receptor antagonist exendin 9-39. Diphtheria toxin-induced knockdown of glucagon producing cells led to reduced somatostatin secretion in response to 12 mmol/l glucose and arginine infusions. In Gcgr-/- mice (where glucagon levels are dramatically increased) overall somatostatin secretion was increased. However, infusion of exendin 9-39 in Gcgr-/- mice completely abolished somatostatin secretion in response to glucose and arginine. Neither insulin nor an insulin receptor antagonist (S961) had any effect on somatostatin secretion. CONCLUSIONS/INTERPRETATION: Our findings demonstrate that somatostatin and glucagon secretion are linked in a reciprocal feedback cycle with somatostatin inhibiting glucagon secretion at low and high glucose levels, and glucagon stimulating somatostatin secretion via the glucagon and GLP-1 receptors. Graphical abstract.
Subject(s)
Glucagon/physiology , Insulin/physiology , Somatostatin/metabolism , Animals , Arginine/administration & dosage , Cell Communication , Diphtheria Toxin/pharmacology , Gene Knockdown Techniques , Glucagon/administration & dosage , Glucagon-Like Peptide-1 Receptor/drug effects , Glucagon-Like Peptide-1 Receptor/physiology , Glucagon-Secreting Cells/drug effects , Glucagon-Secreting Cells/physiology , Glucose/administration & dosage , Insulin/administration & dosage , Insulin Secretion/drug effects , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Glucagon/deficiency , Receptors, Glucagon/genetics , Receptors, Glucagon/physiology , Receptors, Somatostatin/antagonists & inhibitors , Signal Transduction/physiology , Somatostatin/administration & dosageABSTRACT
Adenoviral vectors are superior to plasmid vectors in their gene transport efficiency. The A subunit of the diphtheria toxin (DTA) gene is a popular suicide gene in cancer gene therapy. However, DTA is seldom used in adenoviral therapy due to its great toxicity. The toxicity of DTA is so great that even a single molecule of DTA is enough to kill one cell. To avoid this highly toxic effect on normal cells, DTA should be controlled by tumor-specific promoters. The survivin promoter is a widely used tumor-specific promoter. But genes driven by the survivin promoter show a low level of basal gene expression in non-cancer cells. DTA driven by the survivin promoter in adenoviral vectors may be highly toxic not only to cancer cells but also to normal cells. Therefore, DTA should be attenuated when it is used in adenoviral vectors driven by the survivin promoter. In this study, we compared the three kinds of recombinant adenoviruses that carry DTA or its attenuated forms (DTA176 and DTA197) in the treatment of human lung cancer. The results showed that in comparison with both DTA and DTA176, DTA197 is more suitable for adenoviral cancer therapy controlled by the survivin promoter. In addition, Adsur-DTA197 (DTA197 delivered by an adenoviral vector with the survivin promoter) sensitized human lung cancer cells to cisplatin both in vitro and in vivo. These results indicated that Adsur-DTA197 may be a potential chemosensitizer in cancer therapy.
Subject(s)
Adenoviridae/metabolism , Diphtheria Toxin/therapeutic use , Genetic Vectors/therapeutic use , Lung Neoplasms/drug therapy , Animals , Diphtheria Toxin/pharmacology , Genetic Vectors/pharmacology , Humans , Lung Neoplasms/genetics , Mice , Survivin/metabolismABSTRACT
Enzyme replacement therapy, in which a functional copy of an enzyme is injected either systemically or directly into the brain of affected individuals, has proven to be an effective strategy for treating certain lysosomal storage diseases. The inefficient uptake of recombinant enzymes via the mannose-6-phosphate receptor, however, prohibits the broad utility of replacement therapy. Here, to improve the efficiency and efficacy of lysosomal enzyme uptake, we exploited the strategy used by diphtheria toxin to enter into the endolysosomal network of cells by creating a chimera between the receptor-binding fragment of diphtheria toxin and the lysosomal hydrolase TPP1. We show that chimeric TPP1 binds with high affinity to target cells and is efficiently delivered into lysosomes. Further, we show superior uptake of chimeric TPP1 over TPP1 alone in brain tissue following intracerebroventricular injection in mice lacking TPP1, demonstrating the potential of this strategy for enhancing lysosomal storage disease therapy.
Subject(s)
Diphtheria Toxin , Enzyme Replacement Therapy , Animals , Brain/metabolism , Diphtheria Toxin/metabolism , Diphtheria Toxin/pharmacology , Lysosomes/metabolism , Mice , Receptor, IGF Type 2/genetics , Receptor, IGF Type 2/metabolism , Recombinant Proteins/metabolismABSTRACT
Toxins, while harmful and potentially lethal, have been engineered to develop potent therapeutics including cytotoxins and immunotoxins (ITs), which are modalities with highly selective targeting capabilities. Currently, three cytotoxins and IT are FDA-approved for treatment of multiple forms of hematological cancer, and additional ITs are tested in the clinical trials or at the preclinical level. For next generation of ITs, as well as antibody-mediated drug delivery systems, specific targeting by monoclonal antibodies is critical to enhance efficacies and reduce side effects, and this methodological field remains open to discover potent therapeutic monoclonal antibodies. Here, we describe our application of engineered toxin termed a cell-based IT screening system. This unique screening strategy offers the following advantages: (1) identification of monoclonal antibodies that recognize cell-surface molecules, (2) selection of the antibodies that are internalized into the cells, (3) selection of the antibodies that induce cytotoxicity since they are linked with toxins, and (4) determination of state-specific activities of the antibodies by differential screening under multiple experimental conditions. Since the functional monoclonal antibodies with internalization capacities have been identified successfully, we have pursued their subsequent modifications beyond antibody drug conjugates, resulting in development of immunoliposomes. Collectively, this screening system by using engineered toxin is a versatile platform, which enables straight-forward and rapid selection for discovery of novel functional antibodies.