e15045 Background: Berberine is an alkaloid compound with a structure that is highly similar to that of intercalating agents. It affects numerous cell signaling pathways and is widely studied as potential anticancer drug. It is known that berberine affects cancer cells migration through metalloproteinase-2 inhibition, but this effect was never studied on glioma cells. Anti-migratory drugs are of special interest in brain cancer therapy since glioma's highly invasive nature makes total surgical removal of tumor practically impossible. The aim of the study was to evaluate berberine anti-migratory activity on glioma cells. Methods: Cell migration capacity of T98G and U87MG cell lines, as well as primary glioma cell culture established in our laboratory, was assessed via standard wound healing assay with automated image acquisition and analysis on Lionheart FX (BioTek) cell imager. Prior to assay setting up cell cultures were maintained in DMEM medium with L-glutamine (1 μM) (Gibco) and 10% FBS (Gibco) at 37C0 and 5.0% CO2. Cells were seeded at 250 000 cells per well on 24-well plates and incubated overnight in order to attach to plate bottom. After that a vertical wound was made manually in each well, and berberine was added to experimental wells to final concentration 50 mg/L. Plates with cells were continuously incubated and photographed in cell imager at 37C0 and 5.0% CO2. The extent of cells migration was measured as the percent of wound area decrease after 24 hours of incubation in relation to starting time point. Data are given as: Mean ± 95% confidence interval. Results: In our study we berberine exhibited anti-migratory activity in all cell cultures under study. In rather fast growing primary cell culture wound area decrease was 99.23%±0.62% in control sample and 91.75%±0.28% in experimental sample. The difference was small but significant at p < 0.001 level (df = 30). Popular permanent glioma cell lines T98G and U87MG showed more prominent decrease in studied parameter with higher degree of variance at the same time. In T98G wound area decrease was 71.6%±12.3% in control and 48.8%± 7.6% in experimental samples after 24 hours of cultivation in presence of 50 mg/L berberine. While U87MG demonstrated 60.28%±5.13% and 37.5%± 8.34% wound area decrease accordingly. The obtained difference between control and experimental groups in permanent cell cultures was statistically significant at the 0.05 level (df = 30). Conclusions: Our preliminary research proved berberine to be potent anti-migratory agent in glioma treatment. Further investigations are needed to evaluate its ability to inhibit glioma cell expansion in vivo.
Objective. The aim of this work was to obtain the primary cell lines of brain malignant tumors using the explant method. Materials and methods. Thirteen patients of both sexes, aged 22 to 66, were recruited. The tumor material of the patients was fragmented and placed in flasks with complete nutrient medium for glial tumor cells. Subsequently, the material was photographed at various stages of cultivation, the cell morphology was determined, and the rate of monolayer formation at the zero and first passages was assessed. Results. As a result, thirteen primary human cell lines of glial tumors were obtained: six glioblastoma lines, two glioblastoma lines with anaplastic astrocytoma, one anaplastic oligodendroglioma line, one diffuse astrocytoma line, one oligoastrocytoma line and one diffuse protoplasmic astrocytoma line, one anaplastic astrocytoma line. In the culture of diffuse astrocytoma, there were observed the cells forming a network at the bottom of the flask. In the culture of anaplastic astrocytoma at a confluence of 3050 %, fibroblast-like cells were presented, and at a confluence of 100 %, a monolayer was formed with cells intimately adjacent to each other. In the culture of oligoastrocytoma, both fibroblast-like cells and islets of closely intertwined fusiform cells were observed. The same was typical for the cells of diffuse protoplasmic astrocytoma. Anaplastic oligodendroglioma during the first week of cultivation was represented mainly by round cells with a contrast agent, which subsequently attached and actively proliferated. At a confluence of 3080 %, fibroblast-like cells were observed, and at 100 %, spindle-shaped cells closely adjacent to each other. In cultures of glioblastomas, no specific character of cell growth was revealed: spindle-shaped, fibroblast-like cells and cells with long processes forming a network were encountered. Glioblastoma cultures against the background of anaplastic astrocytoma were represented by a network of cells with long processes. At the zero passage, the rate of formation of a 100 % confluence monolayer ranged from 22 to 85 days. At the first passage, the cells reached a full monolayer within 4 to 25 days. At the zero passage, the longest time among all the samples to form the monolayer with a 100 % confluence needed glioblastoma lines on average 59 days. The shortest time to reach a 100 % confluence was required for cells of diffuse astrocytoma, anaplastic oligodendroglioma and glioblastoma against the background of anaplastic astrocytoma 2224 days. Conclusions. In our work, it was shown that the explant method ensures the production of viable cells of glial tumors and the possibility of their further cultivation.
Secondary plant metabolites are a promising source of anticancer drugs. Of particular interest are substances that can reduce the migration activity of cancer cells as potential anti-metastatic drugs. In present work we study the influence of 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one, 5-(hydroxymethyl)furan-2-carbaldehyde and corinan obtained from plants of the genus Petastes sp. on scratch healing rate of permanent cancer lines PC3, A431, CaCo2, HeLa, and T98G. Cells were grown in DMEM medium supplemented with 10% FBS. To perform a scratch test, cells were planted in an amount of 1,5105 per well of a 24-well plate. After cell adhesion, a vertical scratch was applied to the cell monolayer with a plastic tip, after which the medium containing the test substances at a concentration of 40 M was added. Then, within 48 hours, photographing and determining the scratch area were carried out. The degree of scratch overgrowth was determined as the ratio of the difference between the scratch areas after 48 hours of cultivation and the scratch area at the initial moment, expressed as a percentage. As a result of the experiment, it was shown that 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one downregulates scratch healing rate in cultures A431, HeLa, T98G. The high sensitivity of A431 to corinan was also shown.
Purpose of the study. Testing of new chemotherapeutic agents in translational and biology medicine needs studies on immortalized cell lines. However, such models do not always have the biological properties of a tumor in situ, in contrast to primary cell cultures. Primary cultures of lung cancer cells have biological, morphological and molecular characteristics close or identical to tumor cells in vivo. Obtaining collections of primary lung cancer cell lines is an important task in creating various models for preclinical studies.Materials and methods. The materials are represented by postoperative tumor samples obtained from 25 patients with newly diagnosed lung cancer without prior treatment. The following methods were used to obtain primary cultures: enzymatic dissociation in Hanks' solution with the addition of 300 units/ml collagenase I (Thermo Fisher Scientific, USA), enzymatic dissociation using the Brain Tumor Dissoсiation Kit (Miltenyi Biotec, Germany) and 150 units/ml. ml of collagenase I, as well as the method of explants. The following methods were used to remove fibroblasts: the use of the FibrOut™ system (CHI Scientific, USA), magnetic separation of fibroblasts using Anti-Fibroblast MicroBeads (Miltenyi Biotec, Germany), and cold trypsinization.Results. We have obtained 15 primary lung cancer cell cultures that have passed the zero order passage. In this work, the method of enzymatic dissociation turned out to be the most effective. Incubation of lung tumor samples with collagenase for 1 hour preserves the viability and adhesiveness of the cells. The explant method did not show its effectiveness for long-term cultivation, there was no migration of tumor cells to plastic. Magnetic separation, as a method of removing stromal components of fibroblasts, showed the greatest efficiency, while maintaining the viability of tumor cells.Conclusion. The obtained primary cell cultures of lung cancer can be used for many tasks of experimental oncology: studies of the biological characteristics of lung cancer, development of preclinical models for the studies on new chemotherapeutic drugs.
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