Antitumor efficiencies of monoclonal antibodies to connexin-43 second extracellular loop (MAbE2Cx43), temozolomide, and fractionated γ-irradiation in the monotherapy mode and in several optimized combinations were studied in Wistar rats with induced C6 glioma. The survival of animals with glioma and the dynamics of intracerebral tumor development were evaluated by MRT. Temozolomide monotherapy (200 mg/m(2)) and isolated radiotherapy in a total dose of 36 Gy shifted the survival median from 28 days (no therapy) to 34 and 38 days, respectively; 100% animals died under conditions of temozolomide monotherapy and radiotherapy. Monotherapy with MAbE2Cx43 in a dose of 5 mg/kg led to significant regression of the tumor (according to MRT data), cure of 19.23% animals with glioma, and prolongation of the survival median to 39.5 days after tumor implantation. Combined therapy with MAbE2Cx43 and temozolomide completely abolished the antitumor effect (survival median 29 days). Treatment with MAbE2Cx43 in combination with radiotherapy was associated with mutual boosting of the therapeutic efficiencies, leading to a significant inhibition of tumor development and prolongation of the survival median to 60 days. The mechanism of tumorsuppressive activity of the antibodies could be due to connexon blockade in Cx43-positive glioma cells in the peritumor invasion zone. Higher efficiency of combined therapy was presumably due to the increase in blood-brain barrier permeability for antibodies after irradiation of the brain and to additional inhibitory effect of antibodies towards radioresistant migrating glioma cells. The results suggested that MAbE2Cx43 could be effective as the first-line drug in combined therapy for poorly differentiated gliomas.
We studied the effect of γ-irradiation on HUVEC endothelial cells co-cultured with allogeneic astrocytes. This 2D in vitro model of the blood-brain barrier has the same parameters as cerebral microvascular endothelial cells forming the blood-brain barrier and allows reproducing its functions in vivo. Dose-dependent changes in cell morphology and violation of monolayer integrity were observed. Real-time PCR and immunocytochemical analysis revealed changes in the expression of tight (ZO-1, claudin-5) and adherens junction protein (vascular endothelial cadherin, β-catenin) mRNA. Expression of tight and adherens junction proteins mRNA decreased in 2, 24, and 48 h after irradiation in doses of 2, 4, and 6 Gy. Significant dose-dependent changes were found only for β-catenin mRNA expression in 2 h after exposition. This model of blood-brain barrier in vitro can be used for studying the molecular mechanisms regulating permeability of cerebral endothelium under normal conditions and after pathological exposures, e.g. γ-irradiation.
The flurry of publications devoted to the functions of long non-coding RNAs (lncRNAs) published in the last decade leaves no doubt about the exceptional importance of lncRNAs in various areas including tumor biology. Contribution of lncRNAs to the early stages of oncogenesis remains poorly understood. In this study we explored a new role for lncRNAs: stimulation of driver oncogenic mutations that result from specific chromosomal rearrangements. We demonstrated that lncRNA CASTL1 (ENSG00000269945) stimulates the formation of the CCDC6-RET inversion (RET/PTC1) in human thyroid cells subjected to radiation or chemical DNA damage. Facilitation of chromosomal rearrangement requires lncRNA to contain regions complementary to the introns of both CCDC6 and RET genes as deletion of these regions deprives CASTL1 of the ability to stimulate the gene fusion. We found that CASTL1 expression is elevated in tumors with CCDC6-RET fusion which is the most frequent rearrangement in papillary thyroid carcinoma. Our results open a new venue for the studies of early oncogenesis in various tumor types, especially those associated with physical or chemical DNA damage.
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