ABSTRACT
The effects of Hedgehog signaling inhibitor (cyclopamine) and activator (Shh) on drug resistance of U251-MG human glioma cells and human astrocyte culture to cisplatin, temozolomide, and doxorubicin were studied. Cyclopamine and Shh modified the drug resistance of U251-MG cells but not of human astrocytes. Experiments with cyclopamine, Shh, and chemical drugs can contribute to detection of the mechanisms of signaling effects on the drug resistance processes, while the experimental data can serve as one of the criteria for choosing individual chemotherapy for patients.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Hedgehog Proteins/genetics , Neuroglia/drug effects , Signal Transduction/genetics , Astrocytes/cytology , Astrocytes/drug effects , Astrocytes/metabolism , Cell Differentiation , Cell Line, Tumor , Cells, Cultured , Cisplatin/pharmacology , Dacarbazine/analogs & derivatives , Dacarbazine/pharmacology , Doxorubicin/pharmacology , Hedgehog Proteins/antagonists & inhibitors , Hedgehog Proteins/metabolism , Humans , Neuroglia/metabolism , Neuroglia/pathology , Peptides/pharmacology , Temozolomide , Veratrum Alkaloids/pharmacologyABSTRACT
We studied internalization of vector nanocarriers loaded with plasmid DNA into C6 glioma cells. For improving selectivity of plasmid delivery, the liposomes were conjugated with monoclonal antibodies to VEGF and its receptor VEGFR2. Flow cytofluorometry and laser scanning confocal microscopy showed more intensive (more than 2-fold) internalization and accumulation of antibody-vectorized liposomes in C6 glioma cells in comparison with the control (liposomes conjugated with non-specific antibodies and non-vectorized liposomes). Using quantitative analysis of fluorescent signal, we showed that cationic immunoliposomes significantly more effective delivered pCop-Green-N plasmid DNA and ensured effective transfection of C6 glioma cells.
Subject(s)
Brain Neoplasms/genetics , Glioma/genetics , Glioma/therapy , Liposomes/chemistry , Plasmids/chemistry , Plasmids/genetics , Animals , Brain Neoplasms/therapy , Cell Line, Tumor , Flow Cytometry , Genetic Therapy , Microscopy, Confocal , Rats , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor Receptor-2/geneticsABSTRACT
We studied the effect of an activator (ShTh) and an inhibitor (cyclopamine) of the Hedgehog signaling pathway on proliferation of human glioma cell lines U87-MG and U251-MG and cultured human astrocytes. The Hedgehog signaling pathway is activated in glioma cells, but not in cultured human astrocytes. Experiments with Shh and cyclopamine can serve as an additional criterion for assessing activity of Hedgehog signaling in known cell lines and primary cultured cells.
Subject(s)
Cell Proliferation , Glioblastoma/metabolism , Signal Transduction , Astrocytes/physiology , Cell Line, Tumor , Glioblastoma/pathology , Hedgehog Proteins/agonists , Hedgehog Proteins/metabolism , Humans , Ligands , Veratrum Alkaloids/pharmacologyABSTRACT
We obtained the morphologically, cytofluorometrically, and functionally mature dendritic cells from rats that were pulsed with antigens of the C6 glioma tissue extract. The concentrations of angiogenesis antigens (VEGF, VEGFR-1, and VEGFR-2) and periglioma zone proteins (GFAP, connexin 43, and BSAT1) in the pulsing extract were measured by ELISA. Our results drove us to a conclusion that despite mature phenotype of pulsed dendritic cell, the antigenic composition of glioma tissue extracts should be modified.
Subject(s)
Antigens, Neoplasm/genetics , Brain Neoplasms/chemistry , Complex Mixtures/pharmacology , Dendritic Cells/drug effects , Glioma/chemistry , Animals , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, Neoplasm/immunology , Brain Chemistry , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Complex Mixtures/chemistry , Connexin 43/genetics , Connexin 43/immunology , Dendritic Cells/cytology , Dendritic Cells/immunology , Gene Expression Regulation/immunology , Glial Fibrillary Acidic Protein/genetics , Glial Fibrillary Acidic Protein/immunology , Glioma/genetics , Glioma/metabolism , Glioma/pathology , Humans , Immunophenotyping , Neoplasm Transplantation , Organic Cation Transport Proteins/genetics , Organic Cation Transport Proteins/immunology , Primary Cell Culture , Rats , Stereotaxic Techniques , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/immunology , Vascular Endothelial Growth Factor Receptor-1/genetics , Vascular Endothelial Growth Factor Receptor-1/immunology , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/immunologyABSTRACT
The tumor-suppressive effect of rat mesenchymal stem cells against low-differentiated rat C6 glioma cells during their direct and indirect co-culturing and during culturing of C6 glioma cells in the medium conditioned by mesenchymal stem cells was studied in an in vitro experiment. The most pronounced antitumor activity of mesenchymal stem cells was observed during direct co-culturing with C6 glioma cells. The number of live C6 glioma cells during indirect co-culturing and during culturing in conditioned medium was slightly higher than during direct co-culturing, but significantly differed from the control (C6 glioma cells cultured in medium conditioned by C6 glioma cells). The cytotoxic effect of medium conditioned by mesenchymal stem cells was not related to medium depletion by glioma cells during their growth. The medium conditioned by other "non-stem" cells (rat astrocytes and fibroblasts) produced no tumor-suppressive effect. Rat mesenchymal stem cells, similar to rat C6 glioma cells express connexin 43, the main astroglial gap junction protein. During co-culturing, mesenchymal stem cells and glioma C6 cells formed functionally active gap junctions. Gap junction blockade with connexon inhibitor carbenoxolone attenuated the antitumor effect observed during direct co-culturing of C6 glioma cells and mesenchymal stem cells to the level produced by conditioned medium. Cell-cell signaling mediated by gap junctions can be a mechanism of the tumor-suppressive effect of mesenchymal stem cells against C6 glioma cells. This phenomenon can be used for the development of new methods of cell therapy for high-grade malignant gliomas.
Subject(s)
Brain Neoplasms/pathology , Cell Communication/physiology , Glioma/pathology , Mesenchymal Stem Cells/metabolism , Signal Transduction/physiology , Animals , Antineoplastic Agents , Cell Differentiation , Cell Line, Tumor , Cell Movement , Cell- and Tissue-Based Therapy , Coculture Techniques , Culture Media, Conditioned/pharmacology , Rats , Rats, Wistar , Tight Junctions/physiologyABSTRACT
The review summarizes current knowledge on the Hedgehog signaling pathway, its role in normal embryogenesis and/or initiation and progression of neuro-oncological diseases, especially of high-grade gliomas, the most malignant neuroepithelial tumors. The main proteins forming the Hedgehog signaling pathway include Shh, PTCH1, SMO, HHIP, SUFU and GLI1 isoforms. Effects of other signaling pathways on the family of transcription factors GLI and other proteins are described. The review summarizes modern data about the impact of the Hedgehog signaling pathway on proliferation, migration activity and invasiveness, and also on tumor neoangiogenesis and tumor cell chemoresistance. The role of the Hedgehog signaling pathway in origin of cancer stem cells and epithelial-mesenchymal transition is also analyzed. Some prospects for new anticancer drugs acting on components of the Hedgehog signaling pathway inhibitors are demonstrated.
Subject(s)
Central Nervous System Neoplasms/metabolism , Drug Resistance, Neoplasm , Glioma/metabolism , Hedgehog Proteins/metabolism , Animals , Cell Movement , Cell Proliferation , Central Nervous System Neoplasms/pathology , Glioma/pathology , Hedgehog Proteins/genetics , Humans , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , Zinc Finger Protein GLI1ABSTRACT
The formation of functional gap junctions between mesenchymal stem cells and cells of low-grade rat glioma C6 cells was studied in in vitro experiments. Immunocytochemical analysis with antibodies to connexin 43 extracellular loop 2 showed that mesenchymal stem cells as well as C6 glioma cells express the main astroglial gap junction protein connexin 43. Analysis of migration activity showed that mesenchymal stem cells actively migrate towards C6 glioma cells. During co-culturing, mesenchymal stem cells and glioma C6 form functionally active gap junctions mediating the transport of cytoplasmic dye from glioma cells to mesenchymal stem cells in the opposite direction. Fluorometry showed that the intensity of transport of low-molecular substances through heterologous gap junctions between mesenchymal stem cells and glioma cells is similar to that through homologous gap junctions between glioma cells. This phenomenon can be used for the development of new methods of cell therapy of high-grade gliomas.
Subject(s)
Biological Transport/physiology , Connexin 43/metabolism , Gap Junctions/physiology , Glioma/pathology , Mesenchymal Stem Cells/metabolism , Animals , Cell Communication/physiology , Cell Line, Tumor , Cell- and Tissue-Based Therapy/methods , Coculture Techniques , Connexin 43/biosynthesis , Fluorescent Dyes/metabolism , Fluorometry , Rats , Rats, WistarABSTRACT
A model of highly metastasizing orthotopic allogeneic breast carcinoma was reproduced and standardized in experiments on BALB/c mice. 4T1 cells characterized by high metastatic activity were transfected with red fluorescent protein (RFP) gene or firefly luciferase (Luc2) gene. Unmodified 4T1 cells and modified 4T1-RFP and 4T1-Luc2 cells were subcutaneously injected to mature female mice into the second mammary fat pads. Quantitative evaluation of the primary node and visceral metastases was performed using magnetic-resonance imaging, X-ray and optical tomography. Modification of 4T1 cells with RFP gene considerably reduced their invasive and metastatic potential and led to spontaneous regression of the primary tumor in 20% cases. Modification of 4T1 cells with Luc2 gene had practically no effect on proliferative, invasive, and metastatic characteristics of the tumor and provided the possibility of quantitative analysis of the primary tumor dynamics by the luminescence intensity. The survival median in mice receiving unmodified 4T1 cells and transfected 4T1-RFP and 4Т1-Luc2 cells was 32, 42, and 38 days, respectively. Neither primary node nor tumor metastases accumulated gadolinium-containing contrast agent and Alasens fluorescent tracer. After implantation of 4T1 and 4Т1-Luc2 cells, multiple metastases were more often detected in the lungs, liver, spleen, spine, and regional lymph nodes and less frequently in the brain, which corresponded to metastasizing profile of human breast cancer. The developed model of orthotopic breast carcinoma 4T1 in BALB/c mice with complex detection of multiple organ metastases using X-ray microCT, optical, and MRI can be recommended for preclinical studies of new antitumor preparations.
