Создание ортотопических опухолей в молочной железе мышей BALB/C NUDE клетками рака молочной железы человека MCF-7 и ее VDAC-дефицитными производными

Purpose to study the tumor-forming activity of wild-type MC F-7 cells carrying a full set of porins (VDAC 1, VDAC 2, VDAC 3), as well as their genetically modified cells, from which one of the isoforms was removed (MC F-7 VDAC 1 KO, MC F-7 VDAC 2 KO, MC F -7 VDAC 3 KO).Material and Methods. The study was aimed at establishing of an animal model of orthotopic tumors in the mammary gland of immunodeficient BAL B/c nude mice by implanting a suspension of human breast cancer cells (MC F-7) and derivatives of these cells generated by targeted knockout of one of the selected mitochondrial porin isoforms (VDAC 1, VDAC 2 or VDAC 3). Suspensions of either wild-type MC F-7 cell lines containing all three porin isoforms (VDAC 1, VDAC 2 and VDAC 3) or their VDAC -deficient derivatives (MC F-7 VDAC 1 KO, MC F-7 VDAC 2 KO and MC F-7 VDAC 3 KO) were injected into mammary fat pads of BAL B/c nude mice at a dose of 4x106 cells per injection. A pathomorphological analysis of the place of implantation of tumor cells, the tumor itself, as well as the organs of the abdominal and thoracic cavity was carried out.Results. The study shows the feasibility of successful creation of orthotopic tumors in the adipose tissue of immunodeficient BAL B/c nude mice with MC F-7 human breast cancer epithelial cells containing a complete set of mitochondrial porin isoforms and their VDAC -deficient derivatives. The tumor-forming activity of the implanted cells was shown to correlate with their cytotoxic effect on the internal organs of animals. Pathological analysis showed that all implanted cell cultures, such as MC F-7 WT, MC F-7 VDAC 2 KO and MC F-7 VDAC 3 KO, except for MC F-7 VDAC 1 KO cells, which did not form tumors, caused pathological changes in the lungs, liver and spleen, as well as the presence of other tumor-like lesions.Conclusion. The data obtained will be used to optimize the injection volume and cell number, as well as to refine the dynamics of tumor growth, suitable for studying the effect of anticancer drugs on tumors formed by human breast cancer cells (MC F-7) and its genetically modified VDAC -deficient derivatives.Цель исследования – изучить опухолеобразующую активность «диких» клеток MC F-7, несущих полный набор поринов (VDAC 1, VDAC 2, VDAC 3), а также их генетически модифицированных клеток, из которых удалена одна из изоформ (MC F-7 VDAC 1 KO, MC F-7 VDAC 2 KO, MC F-7 VDAC 3 KO).Материал и методы. Исследование направлено на создание животной модели ортотопических опухолей в молочной железе иммунодефицитных мышей BAL B/c Nude путем имплантации суспензии клеток рака молочной железы человека (MC F-7) и производных этих клеток, полученных нокаутом одной из выбранных изоформ митохондриальных поринов (VDAC 1, VDAC 2 или VDAC 3). Опухоли создавались путем инъекции в жировую ткань молочной железы мышей BAL B/c Nude суспензии клеточных линий либо диких MC F-7 (содержащих все три изоформы поринов, VDAC 1, VDAC 2 и VDAC 3) либо ее VDAC -дефицитных производных «MC F-7 VDAC 1 KO», «MC F-7 VDAC 2 KO» и «MC F-7 VDAC 3 KO» в дозе 4х106 клеток на одну инъекцию. Проведён патоморфологический анализ места имплантации опухолевых клеток, самой опухоли, а также органов брюшной и грудной полости.Результаты. Показана возможность успешного создания ортотопических опухолей в жировой ткани иммунодефицитных голых мышей BAL B/c Nude эпителиальными клетками рака молочной железы человека «MC F-7 WT», содержащими полный набор изоформ митохондриальных поринов и ее VDAC -дефицитными производными. Опухолеобразующая активность имплантированных клеток коррелирует с их цитотоксическим действием на внутренние органы животного. По результатам патоморфологического анализа можно сделать вывод о том, что за исключением клеток типа «MC F-7 VDAC 1 KO», которые не образовали опухолей, все остальные имплантированные клеточные культуры «MC F-7 WT», «MC F-7 VDAC 2 KO» и «MC F-7 VDAC 3 KO» вызывали патологические изменения состояния легких, печени и селезенки, а также наличие других опухолевидных новообразований.Заключение. Полученные данные будут использованы для оптимизации объема инъекции и количества клеток, а также для уточнения динамики роста опухолей, пригодного для изучения действия противоопухолевых препаратов на опухолях, образованных клетками рака молочной железы человека (MC F-7) и ее генетически модифицированными VDAC -дефицитными производными

Diazoxide attenuates autoimmune encephalomyelitis and modulates lymphocyte proliferation and dendritic cell functionality

Activation of mitochondrial ATP-sensitive potassium (KATP) channels is postulated as an effective mechanism to confer cardio and neuroprotection, especially in situations associated to oxidative stress. Pharmacological activation of these channels inhibits glia-mediated neuroinflammation. In this way, diazoxide, an old-known mitochondrial KATP channel opener, has been proposed as an effective and safe treatment for different neurodegenerative diseases, demonstrating efficacy in different animal models, including the experimental autoimmune encephalomyelitis (EAE), an animal model for Multiple Sclerosis. Although neuroprotection and modulation of glial reactivity could alone explain the positive effects of diazoxide administration in EAE mice, little is known of its effects on the immune system and the autoimmune reaction that triggers the EAE pathology. The aim of the present work was to study the effects of diazoxide in autoimmune key processes related with EAE, such as antigen presentation and lymphocyte activation and proliferation. Results show that, although diazoxide treatment inhibited in vitro and ex-vivo lymphocyte proliferation from whole splenocytes it had no effect in isolated CD4(+) T cells. In any case, treatment had no impact in lymphocyte activation. Diazoxide can also slightly decrease CD83, CD80, CD86 and major histocompatibility complex class II expression in cultured dendritic cells, demonstrating a possible role in modulating antigen presentation. Taken together, our results indicate that diazoxide treatment attenuates autoimmune encephalomyelitis pathology without immunosuppressive effect

ATP-sensitive K+ channel openers prevent Ca2+ overload in rat cardiac mitochondria

Mitochondrial dysfunction, secondary to excessive accumulation of Ca2+, has been implicated in cardiac injury. We here examined the action of potassium channel openers on mitochondrial Ca2+ homeostasis, as these cardioprotective ion channel modulators have recently been shown to target a mitochondrial ATP-sensitive K+ channel.In isolated cardiac mitochondria, diazoxide and pinacidil decreased the rate and magnitude of Ca2+ uptake into the mitochondrial matrix with an IC50 of 65 and 128 μm, respectively. At all stages of Ca2+ uptake, the potassium channel openers depolarized the mitochondrial membrane thereby reducing Ca2+ influx through the potential-dependent mitochondrial uniporter.Diazoxide and pinacidil, in a concentration-dependent manner, also activated release of Ca2+ from mitochondria. This was prevented by cyclosporin A, an inhibitor of Ca2+ release through the mitochondrial permeability transition pore.Replacement of extramitochondrial K+ with mannitol abolished the effects of diazoxide and pinacidil on mitochondrial Ca2+, while the K+ ionophore valinomycin mimicked the effects of the potassium channel openers.ATP and ADP, which block K+ flux through mitochondrial ATP-sensitive K+ channels, inhibited the effects of potassium channel openers, without preventing the action of valinomycin.In intact cardiomyocytes, diazoxide also induced mitochondrial depolarization and decreased mitochondrial Ca2+ content. These effects were inhibited by the mitochondrial ATP-sensitive K+ channel blocker 5-hydroxydecanoic acid.Thus, potassium channel openers prevent mitochondrial Ca2+ overload by reducing the driving force for Ca2+ uptake and by activating cyclosporin-sensitive Ca2+ release. In this regard, modulators of an ATP-sensitive mitochondrial K+ conductance may contribute to the maintenance of mitochondrial Ca2+ homeostasis

Knockout of Slc25a19 causes mitochondrial thiamine pyrophosphate depletion, embryonic lethality, CNS malformations, and anemia

SLC25A19 mutations cause Amish lethal microcephaly (MCPHA), which markedly retards brain development and leads to α-ketoglutaric aciduria. Previous data suggested that SLC25A19, also called DNC, is a mitochondrial deoxyribonucleotide transporter. We generated a knockout mouse model of Slc25a19. These animals had 100% prenatal lethality by embryonic day 12. Affected embryos at embryonic day 10.5 have a neural-tube closure defect with ruffling of the neural fold ridges, a yolk sac erythropoietic failure, and elevated α-ketoglutarate in the amniotic fluid. We found that these animals have normal mitochondrial ribo- and deoxyribonucleoside triphosphate levels, suggesting that transport of these molecules is not the primary role of SLC25A19. We identified thiamine pyrophosphate (ThPP) transport as a candidate function of SLC25A19 through homology searching and confirmed it by using transport assays of the recombinant reconstituted protein. The mitochondria of Slc25a19(−/−) and MCPHA cells have undetectable and markedly reduced ThPP content, respectively. The reduction of ThPP levels causes dysfunction of the α-ketoglutarate dehydrogenase complex, which explains the high levels of this organic acid in MCPHA and suggests that mitochondrial ThPP transport is important for CNS development