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1.
Sci Rep ; 13(1): 20648, 2023 11 24.
Article in English | MEDLINE | ID: mdl-38001360

ABSTRACT

Molecular radiotherapy combines the advantages of systemic administration of highly specific antibodies or peptides and the localized potency of ionizing radiation. A potential target for molecular radiotherapy is the cell surface antigen CD44v6, which is overexpressed in numerous cancers, with limited expression in normal tissues. The aim of the present study was to generate and characterize a panel of human anti-CD44v6 antibodies and identify a suitable candidate for future use in molecular radiotherapy of CD44v6-expressing cancers. Binders were first isolated from large synthetic phage display libraries containing human scFv and Fab antibody fragments. The antibodies were extensively analyzed through in vitro investigations of binding kinetics, affinity, off-target binding, and cell binding. Lead candidates were further subjected to in vivo biodistribution studies in mice bearing anaplastic thyroid cancer xenografts that express high levels of CD44v6. Additionally, antigen-dependent tumor uptake of the lead candidate was verified in additional xenograft models with varying levels of target expression. Interestingly, although only small differences were observed among the top antibody candidates in vitro, significant differences in tumor uptake and retention were uncovered in in vivo experiments. A high-affinity anti-CD44v6 lead drug candidate was identified, mAb UU-40, which exhibited favorable target binding properties and in vivo distribution. In conclusion, a panel of human anti-CD44v6 antibodies was successfully generated and characterized in this study. Through comprehensive evaluation, mAb UU-40 was identified as a promising lead candidate for future molecular radiotherapy of CD44v6-expressing cancers due to its high affinity, excellent target binding properties, and desirable in vivo distribution characteristics.


Subject(s)
Neoplasms , Humans , Animals , Mice , Tissue Distribution , Neoplasms/pathology , Antibodies, Monoclonal/metabolism , Cell Line, Tumor
2.
Psychopharmacology (Berl) ; 232(24): 4421-31, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26407601

ABSTRACT

RATIONALE: Adolescent binge drinking is common and associated with increased risk of substance use disorders. Transition from recreational to habitual ethanol consumption involves alterations in dorsal striatal function, but the long-term impact of adolescent ethanol exposure upon this region remains unclear. OBJECTIVES: This study aimed to characterise and describe relationships between adolescent ethanol exposure, amphetamine self-administration and adult dopamine dynamics in dorsal striatum, including response to amphetamine challenge, in male Wistar rats. METHODS: Ethanol (2 g/kg) or water was administered intragastrically in an episodic binge-like regimen (three continuous days/week) between 4 and 9 weeks of age (i.e. post-natal days 28-59). In adulthood, animals were divided into two groups. In the first, dorsal striatal potassium-evoked dopamine release was examined via chronoamperometry, in the basal state and after a single amphetamine challenge (2 mg/kg, i.v.). In the second, amphetamine self-administration behaviour was studied (i.e. fixed and progressive ratio) before chronoamperometric analysis was conducted as described above. RESULTS: Adolescent ethanol exposure suppressed locally evoked dopamine response after amphetamine challenge in adulthood, whereas in the basal state, no differences in dopamine dynamics were detected. Ethanol-exposed animals showed no differences in adult amphetamine self-administration behaviour but an abolished effect on dopamine removal in response to a single amphetamine challenge after self-administration. CONCLUSION: Amphetamine challenges in adult rats revealed differences in in vivo dopamine function after adolescent ethanol exposure. The attenuated drug response in ethanol-exposed animals may affect habit formation and contribute to increased risk for substance use disorders as a consequence of adolescent ethanol.


Subject(s)
Amphetamine/administration & dosage , Corpus Striatum/drug effects , Dopamine Uptake Inhibitors/administration & dosage , Dopamine/metabolism , Ethanol/pharmacology , Animals , Corpus Striatum/metabolism , Male , Rats , Rats, Wistar , Self Administration
3.
Cell Death Dis ; 3: e298, 2012 Apr 19.
Article in English | MEDLINE | ID: mdl-22513872

ABSTRACT

Wig-1 is a transcriptional target of the p53 tumor suppressor and encodes an mRNA stability-regulating protein. We show here that Wig-1 knockdown causes a dramatic inhibition of N-Myc expression and triggers differentiation in neuroblastoma cells carrying amplified N-Myc. Transient Wig-1 knockdown significantly delays development of N-Myc-driven tumors in mice. We also show that N-Myc expression is induced upon moderate p53-activating stress, suggesting a role of the p53-Wig-1-N-Myc axis in promoting cell cycle re-entry upon p53-induced cell cycle arrest and DNA repair. Moreover, our findings raise possibilities for the improved treatment of poor prognosis neuroblastomas that carry amplified N-Myc.


Subject(s)
Carrier Proteins/metabolism , Neoplasms/pathology , Nuclear Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , RNA, Messenger/metabolism , Animals , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Cell Cycle Checkpoints , Cell Line, Tumor , DNA Repair , Female , HCT116 Cells , Humans , Mice , Mice, Nude , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Proto-Oncogene Proteins c-myc/genetics , RNA Interference , RNA, Small Interfering/metabolism , RNA-Binding Proteins , Transplantation, Heterologous , Tumor Suppressor Protein p53/metabolism
4.
Br J Cancer ; 106(11): 1807-15, 2012 May 22.
Article in English | MEDLINE | ID: mdl-22531632

ABSTRACT

BACKGROUND: Cellular quiescence is a state of reversible proliferation arrest that is induced by anti-mitogenic signals. The endogenous cardiac glycoside ouabain is a specific ligand of the ubiquitous sodium pump, Na,K-ATPase, also known to regulate cell growth through unknown signalling pathways. METHODS: To investigate the role of ouabain/Na,K-ATPase in uncontrolled neuroblastoma growth we used xenografts, flow cytometry, immunostaining, comet assay, real-time PCR, and electrophysiology after various treatment strategies. RESULTS: The ouabain/Na,K-ATPase complex induced quiescence in malignant neuroblastoma. Tumour growth was reduced by >50% when neuroblastoma cells were xenografted into immune-deficient mice that were fed with ouabain. Ouabain-induced S-G2 phase arrest, activated the DNA-damage response (DDR) pathway marker γH2AX, increased the cell cycle regulator p21(Waf1/Cip1) and upregulated the quiescence-specific transcription factor hairy and enhancer of split1 (HES1), causing neuroblastoma cells to ultimately enter G0. Cells re-entered the cell cycle and resumed proliferation, without showing DNA damage, when ouabain was removed. CONCLUSION: These findings demonstrate a novel action of ouabain/Na,K-ATPase as a regulator of quiescence in neuroblastoma, suggesting that ouabain can be used in chemotherapies to suppress tumour growth and/or arrest cells to increase the therapeutic index in combination therapies.


Subject(s)
Histones/metabolism , Neuroblastoma/metabolism , Ouabain/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Comet Assay , Female , Flow Cytometry , Humans , Mice , Mice, Nude , Ouabain/pharmacology , Real-Time Polymerase Chain Reaction , Transplantation, Heterologous
5.
Oncogene ; 27(20): 2910-22, 2008 May 01.
Article in English | MEDLINE | ID: mdl-18026138

ABSTRACT

Mammalian target of rapamycin (mTOR) has been shown to play an important function in cell proliferation, metabolism and tumorigenesis, and proteins that regulate signaling through mTOR are frequently altered in human cancers. In this study we investigated the phosphorylation status of key proteins in the PI3K/AKT/mTOR pathway and the effects of the mTOR inhibitors rapamycin and CCI-779 on neuroblastoma tumorigenesis. Significant expression of activated AKT and mTOR were detected in all primary neuroblastoma tissue samples investigated, but not in non-malignant adrenal medullas. mTOR inhibitors showed antiproliferative effects on neuroblastoma cells in vitro. Neuroblastoma cell lines expressing high levels of MYCN were significantly more sensitive to mTOR inhibitors compared to cell lines expressing low MYCN levels. Established neuroblastoma tumors treated with mTOR inhibitors in vivo showed increased apoptosis, decreased proliferation and inhibition of angiogenesis. Importantly, mTOR inhibitors induced downregulation of vascular endothelial growth factor A (VEGF-A) secretion, cyclin D1 and MYCN protein expression in vitro and in vivo. Our data suggest that mTOR inhibitors have therapeutic efficacy on aggressive MYCN amplified neuroblastomas.


Subject(s)
Antibiotics, Antineoplastic/pharmacology , Down-Regulation/drug effects , Growth Inhibitors/pharmacology , Neuroblastoma/pathology , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Oncogene Proteins/antagonists & inhibitors , Oncogene Proteins/genetics , Protein Kinases/metabolism , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Mice , Mice, Nude , N-Myc Proto-Oncogene Protein , Neuroblastoma/drug therapy , Neuroblastoma/metabolism , Nuclear Proteins/biosynthesis , Oncogene Proteins/biosynthesis , Phosphatidylinositol 3-Kinases/physiology , Protein Kinases/biosynthesis , Protein Kinases/genetics , Proto-Oncogene Proteins c-akt/biosynthesis , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/physiology , Signal Transduction/drug effects , Signal Transduction/genetics , Sirolimus/pharmacology , TOR Serine-Threonine Kinases
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