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0THE INFLUENSE OF OVEREXPRESSION OF KLOTHO ON THE CHARACTERISTICS OF GROWTH OF HUMAN GLIOMA CELL CULTURE
Melekhin V. V.1
Institute of Medical Cell Technologies, Ekaterinburg, Russian Federation
Ponomarev A.I.1'2
Institute of Medical Cell Technologies, Ekaterinburg, Russian Federation Ural State Medical University, Ekaterinburg, Russian Federation
Kostyukova S.V.1'2
Institute of Medical Cell Technologies, Ekaterinburg, Russian Federation Ural State Medical University, Ekaterinburg, Russian Federation
Satonkina O.A.1'2
Institute of Medical Cell Technologies, Ekaterinburg, Russian Federation Ural State Medical University, Ekaterinburg, Russian Federation
Makeev O.G.1'2
Institute of Medical Cell Technologies, Ekaterinburg, Russian Federation Ural State Medical University, Ekaterinburg, Russian Federation
Abstract
This article represents the results of the research conducted on cell line culture of human glioma A-172. These cells induced overexpression of Klotho gene, which had an input on viability of cells. Valid decrease of the research parameters of tumor cells been registered under the influence of Klotho gene overexpression. Keywords: Klotho, glioma, A-172.
The Klotho gene, identified back in 1997 [7] as an anti-aging gene, is currently being actively studied as a tumor suppressor. A number of studies have demonstrated that overexpression of a gene suppresses proliferation and induces apoptosis of tumor cells of various lines. Thus, positive results were obtained when studying the effect of Klotho on the culture of breast cancer cells [5], lung cancer [1], and others. This study continue our work, in which it is shown that Klotho overexpression inhibits the proliferative activity in human rhabdomyosarcoma cell culture, and also reduces the concentration of nucleic acids in cells and the intensity of their synthesis [9].
The aim of the study was to evaluate the effect of the non-viral vector-induced overexpression of the secreted form of the Klotho gene on the survival rate in cell culture of human glioma line A-172.
Materials and methods
Studies were performed on a culture of human gli-oma cells line A-172 [6], having ectodermal origin, cultured in DMEM / HamF-12 (SigmaAldrich, USA) containing 10% bovine fetal serum (SigmaAldrich, USA). Incubation conditions: 5% CO2, 37 ° C and 95% humidity. The studies included two groups: experimental and control. In the experimental group, Klotho gene overexpression was simulated by transfection with a plasmid with the gene of the secreted form of the Klotho protein, the control group was left without genetic correction. Plasmid using a kit (zymoresearch, D4015) was isolated from the culture of E. coli, provided by the laboratory of Dr. Hal Dietz of Johns Hopkins University (USA) under an inter-university cooperation agreement. For transfection, Escort III complex of polycationic lipids (SigmaAldrich, USA) was used. The ratio of DNA to lipids in the transfection mixture: 1 pg per 1 pl, respectively. The control group was exposed to polycationic lipids at the same concentration, but without DNA. The culture was planted on three 96-well plates (Orange, Belgium) and incubated for 12 hours. After that, they were subjected to liposomal transfection with a plasmid with the useful gene of the
secreted form of Klotho. Transfected cultures were incubated for 8 hours, then the medium in the culture flasks was changed to standard growth and placed in the incubator for 24, 48 and 72 hours. Next, the plates were removed for the MTT test. This test was carried out using a commercial kit (TOX1-1KT, SigmaAldrich, USA) in accordance with the manufacturer's recommendations.
During work, the culture medium was removed from the wells of the A-172 cells, and then 20 ^l of dye (M5655, SigmaAldrich, USA) was added to each well and returned to the incubator for 4 hours. After this time, a lysis solution (M8910, SigmaAldrich, USA) with a volume of 200 ^l was added to the wells, and then pipetted until the dye crystals were completely dissolved. Next, on the vertical spectrophotometer (Mul-tiskan GO, Thermo Scientific, Finland), the optical density of the obtained solutions was estimated at wavelengths: 570 and 690 nm. The result was determined as the difference in optical density. The number of wells with surviving cells in which the results obtained for the difference in optical density exceeded the threshold value of 0.1 (OD570-690) was evaluated.
All obtained data were subjected to statistical processing on the RStudio program (Version0.99.491 -RStudio, Inc.). Significant differences were confirmed by Fisher's exact test. A value of p <0.05 was considered statistically significant.
Results and discussion
In the first 24 hours there was no significant difference between the control and experimental groups (Table 1). However, after 48 hours, statistically significant differences between the studied groups were found. Thus, the number of viable cell cultures in the experimental and control groups - 8 and 19, non-viable - 22 and 11, respectively (p <0.01). 72 hours after transfection, it turned out that there are only two viable cultures in the experimental group, 15 in the control group, 30 and 18 non-viable in the transfected and control groups, respectively (p <0.001). Thus, in accordance with the obtained results, we can conclude that in the experimental group, compared with the control group,
the viability of cultures subjected to the induction of Klotho overexpression was significantly reduced.
It is also important that in the experimental group the cell viability is significantly reduced over time. And if 48 hours after transfection the number of viable and
Thus, induced Klotho overexpression contributed to a decrease in the survival of cell cultures in the experimental group compared with the control group.
The results obtained by us are evidenced by the literature data. For example, in the lung cancer cell line A-549, it was shown that induced overexpression of the Klotho gene inhibits cell proliferation and stimulates apoptosis. In this regard, it was suggested that the effect of Klotho on Bax and Bcl-2 - genes that affect the apop-totic mechanisms in the cell [2]. The involvement of the Wnt-TCF / beta-catenin signaling pathway in klotho-induced suppression of tumor growth has also been shown [4,8]. It is noteworthy that signaling pathways that are affected by Klotho as a tumor suppressor are common in the development of many cancers. Taking into account the above, it can also be assumed that a significant effect of Klotho gene expression products is realized through signaling pathways, in particular Wnt-TCF / beta-catenin, which explains the different effects of Klotho proteins on normal and tumor cells, the level of which in the latter is significantly lower than normal [2].
It is also possible that the non-canonical Wnt 5A protein can be directly associated with the antitumor effect of Klotho, a high level of which not only determines the aggressive course of a malignant disease, but also contributes to an increase in metastasis and increased activity of tumor cells, as was shown by the example of melanoma [3]
Conclusion:
1. Hyper-expression of the Klotho gene reduces the viability of human glioma cells.
2. The decrease in the viability of the A-172 human glioma cell culture can be due to inhibition of cell proliferation and stimulation of apoptosis.
3. The study of the mechanisms of action of Klotho on the body, both in normal and in pathological conditions, can to a large extent provide a fundamentally new approach to the diagnosis and treatment of oncological diseases.
References
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non-viable in the control group compared to the experimental one does not have a statistically significant difference, then statistically significant differences of 48 and 72 hours in the number of viable cultures of the experimental group indicate a decrease in cell viability.
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Table 1
The number of viable (OD570-690> 0, 1) and non-viable (OD570-690 <0, 1) cultures in the experimental
Ex perimental Control
Viable Nonviable Viable Nonviable
24 hours 2 28 1 29
48 hours 8** 22** 19 11
72 hours 2*** 30*** 15 18
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