Apoptosis indices in liver cell mitochondria in B16/F10 melanoma growing in presence of chronic neurogenic pain

Введение. Печень по количеству, плотности митохондрий один из самых богатых органов, который также является критическим местом для множества метаболических путей. Цель исследования - изучение показателей апоптоза в митохондриях печени самок мышей линии С57ВL/6 при самостоятельном росте меланомы В16/F10 и на фоне коморбидной патологии - хронической нейрогенной боли. Методика. В эксперименте использовали мышей-самок (n=168) линии С57ВL/6. Группы: интактная (n=21); контрольная (n=21) - создание модели хронической нейрогенной боли (ХНБ), путем двусторонней перевязки седалищных нервов; группа сравнения (n=63) - подкожная трансплантация меланомы B16/F10; основная группа (ХНБ+B16/F10) (n=63) - подкожная трансплантация меланомы В16/F10 через 3 нед после моделировия ХНБ. В митохондриях печени методом ИФА определяли концентрацию: цитохрома С (нг/г белка), каспазы 9 (нг/г белка), Bcl-2 (нг/г белка), AIF (нг/г белка), кальция (Са 2+) (мМоль/г белка). Результаты. В митохондриях клеток печени через 1 нед роста меланомы относительно интактных значений фиксировали нарастание уровней AIF в 2,2 раза, цитохрома С в 1,7 раза (р<0,05) и снижение каспазы 9 в 2,0 раза; через 3 нед - падение кальция в 4,7 раза, AIF в 7,1 раза и цитохрома С в 1,7 раза (р<0,05) и накопление каспазы 9 - 1,6 раза (р<0,05). Развитие опухоли при ХНБ через 1 нед сопровождалось уменьшением концентрации AIF в 29,3 раза и цитохрома С в 2,0 раза по сравнению с контрольными значениями (ХНБ). Через 3 недели роста меланомы на фоне ХНБ фиксировали снижение уровней AIF в 6,6 раза, цитохрома С в 4,7 раза и кальция в 32,8 раза, уровень каспазы 9, напротив, повышался в 1,5 раза (р<0,05). Заключение. Наличие коморбидной патологии - ХНБ при опухолевом процессе способствует раннему возникновению нарушений в электронно-транспортной цепи митохондрий клеток печени. Background. The liver is one of the richest organs in terms of the number and density of mitochondria; it is also a critical site for many metabolic pathways. The aim of the study was to analyze indicators of apoptosis in liver mitochondria in female С57ВL/6 mice with B16/F10 melanoma growing alone and in presence of chronic neurogenic pain. Methods. Female С57ВL/6 mice (n=168) were studied. Animals were divided into groups: intact group (n=21); controls (n=21) with a model of chronic neurogenic pain (CNP) created by bilateral sciatic nerve ligation; comparison group (n=63) with subcutaneous transplantation of B16/F10 melanoma; main group (CNP+B16/F10) (n=63) with subcutaneous transplantation of B16/F10 melanoma 3 wks after modeling CNP. Cytochrome C (ng/g protein), caspase-9 (ng/g protein), Bcl-2 (ng/g protein), AIF (ng/g protein), and calcium (Ca2+) (mmol/g protein) were measured by ELISA in the liver mitochondrial fraction. Results. After 1 wk of melanoma growth, AIF increased by 2.2 times, cytochrome C increased by 1.7 times (p<0.05), and caspase-9 decreased by 2.0 times compared to the intact group values. After 3 wks, calcium decreased by 4.7 times, AIF by 7.1 times, cytochrome C by 1.7 times (p<0.05), and caspase-9 increased by 1.6 times (p<0.05). After 1 wk, tumor development in the presence of CNP was accompanied by decreases in AIF by 29.3 times and cytochrome C by 2.0 times, compared to control CNP values. After 3 wks of melanoma growth in presence of CNP, AIF decreased by 6.6 times, cytochrome C by 4.7 times, and calcium by 32.8 times. Caspase-9, on the contrary, increased by 1.5 times (p<0.05). Conclusions. The presence of CNP comorbidity during the tumor development facilitates earlier occurrence of disorders in the electron transport chain of hepatocyte mitochondria.

Noninvasive Tracking of mPEG-poly(Ala) Hydrogel-Embedded MIN6 Cells after Subcutaneous Transplantation in Mice

Recently, we demonstrated the feasibility of subcutaneous transplantation of MIN6 cells embedded in a scaffold with poly(ethylene glycol) methyl ether (mPEG)-poly(Ala) hydrogels. In this study, we further tracked these grafts using magnetic resonance (MR) and bioluminescence imaging. After being incubated overnight with chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles and then mixed with mPEG-poly(Ala) hydrogels, MIN6 cells appeared as dark spots on MR scans. For in vivo experiments, we transfected MIN6 cells with luciferase and/or incubated them overnight with CSPIO overnight; 5 × 106 MIN6 cells embedded in mPEG-poly(Ala) hydrogels were transplanted into the subcutaneous space of each nude mouse. The graft of CSPIO-labeled MIN6 cells was visualized as a distinct hypointense area on MR images located at the implantation site before day 21. However, this area became hyperintense on MR scans for up to 64 days. In addition, positive bioluminescence images were also observed for up to 64 days after transplantation. The histology of removed grafts showed positive insulin and iron staining. These results indicate mPEG-poly(Ala) is a suitable scaffold for β-cell encapsulation and transplantation. Moreover, MR and bioluminescence imaging are useful noninvasive tools for detecting and monitoring mPEG-poly(Ala) hydrogel-embedded MIN6 cells at a subcutaneous site.

Using ultrasound-targeted microbubble destruction to enhance radiotherapy of glioblastoma

Objective To investigate the efficacy and mechanism of ultrasound-targeted microbubble destruction (UTMD) combined with radiotherapy (XRT) on glioblastoma. Methods The enhanced radiosensitization by UTMD was assessed through colony formation and cell apoptosis in Human glioblastoma cells (U87MG). Subcutaneous transplantation tumors in 24 nude mice implanted with U87MG cells were randomly assigned to 4 different treatment groups (Control, UTMD, XRT, and UTMD + XRT) based on tumor sizes (100–300 mm3). Tumor growth was observed for 10 days after treatment, and then histopathology stains (HE, CD34, and γH2AX) were applied to the tumor samples. A TUNEL staining experiment was applied to detect the apoptosis rate of mice tumor samples. Meanwhile, tissue proteins were extracted from animal specimens, and the expressions of dsDNA break repair-related proteins from animal specimens were examined by the western blot. Results When the radiotherapy dose was 4 Gy, the colony formation rate of U87MG cells in the UTMD + XRT group was 32 ± 8%, lower than the XRT group (54 ± 14%, p < 0.01). The early apoptotic rate of the UTMD + XRT group was 21.1 ± 3% at 48 h, higher than that of the XRT group (15.2 ± 4%). The tumor growth curve indicated that the tumor growth was inhibited in the UTMD + XRT group compared with other groups during 10 days of observation. In TUNEL experiment, the apoptotic cells of the UTMD + XRT group were higher than that of the XRT group (p < 0.05). The UTMD + XRT group had the lowest MVD value, but was not significantly different from other groups (p > 0.05). In addition, γH2AX increased due to the addition of UTMD to radiotherapy compared to XRT in immunohistochemistry (p < 0.05). Conclusions Our study clearly demonstrated the enhanced destructive effect of UTMD combined with 4 Gy radiotherapy on glioblastoma. This could be partly achieved by the increased ability of DNA damage of tumor cells.

Xenograft heterotopic modeling of colorectal cancer of various cell types in athymic BALB/c nude mice

Введение. Высокий уровень заболеваемости колоректальным раком стимулирует поиск методов его своевременной диагностики и эффективной терапии, что, в свою очередь, предполагает использование адекватных животных моделей на стадиях разработки и доклинических исследований. Методика. Для моделирования гетеротопических ксенографтов были использованы клеточные культуры колоректального рака человека линий DLD-1, HCT 116 и HT-29. В качестве носителей опухолевых моделей были взяты мыши линии BALB/c nude. Суспензию клеток вводили подкожно в область лопатки с помощью шприца. Результаты. Полученные из Американской коллекции типовых культур (ATCC) клетки культивировали до достижения необходимого количества для моделирования ксенографтов (20 сут). Клетки линии HCT-116 культивировались активнее и образовывали монослой в 3 раза быстрее, чем клетки линии DLD-1 и HT-29. Выживаемость мышей после проведения процедуры подкожной ксенотрансплантации составляла 100%. Образование ксенографтов объемом около 1 см3 наблюдали на 17-е - 26-е сут после прививания клеток. Приживаемость введённых клеток составила 100% для линии HCT-116. Для клеток колоректального рака человека линий DLD-1 и HT-29 приживаемость составила 75% и 60%, соответственно. Заключение. При экспериментальном моделировании подкожных гетеротопических ксенографтов колоректального рака человека на мышах линии BALB/c nude была получена высокая степень воспроизводимости при 100%-ной выживаемости животных-носителей. Introduction. The high prevalence of colorectal cancer (СС) stimulates scientists and physicians to search methods for СС diagnosis and effective therapy, which requires appropriate animal models at the stage of development and preclinical studies. Methods. CC cell cultures (DLD-1, HCT 116, and HT-29) were used for heterotopic xenograft modeling in BALB/c nude mice. The cell suspension was injected subcutaneously with a syringe. Results. CC cells from ATCC (American Type Culture Collection) were cultivated until obtaining the required number of cells for xenograft modeling (20 days). The HCT-116 cell culture developed more actively and formed a monolayer three times faster than DLD-1 and HT-29 cells. Survival of the mice after subcutaneous transplantation was 100%. Xenografts of approximately 1 cm3 volume formed at 17-26 days after grafting. Transplantability of the injected cells was 100% for HCT-116, 75% for DLD-1, and 60% for HT-29. Conclusion. The experimental modeling of subcutaneous heterotopic CC xenografts in BALB/c nude mice showed a high level of reproducibility with absolute 100% - survival of recipient mice.

Co-Microencapsulation of Islets and MSC CellSaics, Mosaic-Like Aggregates of MSCs and Recombinant Peptide Pieces, and Therapeutic Effects of Their Subcutaneous Transplantation on Diabetes

The subcutaneous transplantation of microencapsulated islets has been extensively studied as a therapeutic approach for type I diabetes. However, due to the lower vascular density and strong inflammatory response in the subcutaneous area, there have been few reports of successfully normalized blood glucose levels. To address this issue, we developed mosaic-like aggregates comprised of mesenchymal stem cells (MSCs) and recombinant peptide pieces called MSC CellSaics, which provide a continuous release of angiogenic factors and anti-inflammatory cytokines. Our previous report revealed that the diabetes of immunodeficient diabetic model mice was reversed by the subcutaneous co-transplantation of the MSC CellSaics and rat islets. In this study, we focused on the development of immune-isolating microcapsules to co-encapsulate the MSC CellSaics and rat islets, and their therapeutic efficiency via subcutaneous transplantation into immunocompetent diabetic model mice. As blood glucose level was monitored for 28 days following transplantation, the normalization rate of the new immuno-isolating microcapsules was confirmed to be significantly higher than those of the microcapsules without the MSC CellSaics, and the MSC CellSaics transplanted outside the microcapsules (p < 0.01). Furthermore, the number of islets required for the treatment was reduced. In the stained sections, a larger number/area of blood vessels was observed around the new immuno-isolating microcapsules, which suggests that angiogenic factors secreted by the MSC CellSaics through the microcapsules function locally for their enhanced efficacy.