Antiproliferative effect of L1CAM-specific aptamers in human glioblastoma cell cultures

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Abstract

Glioblastoma remains an uncurable form of brain tumor. Existing methods of therapy allows to insignificantly prolong patient’s lifespan with this diagnosis. Thus, it is necessary to search for new approaches and develop new principals of glioblastoma therapy. In this paper, we describe the principle of impact on glioblastoma tumor cells, which consists in targeted inhibition of the proliferation of L1CAM-positive cells using aptamers. L1CAM is considered to be a marker of tumor glioma stem cells, the presence of which in a tumor may be responsible for resistance to therapy. As a result of the work, the yly12 aptamer was selected from a panel of aptamers for L1CAM and its antiproliferative effect was shown, which was more pronounced on human glioblastoma cells with increased expression of L1CAM. Thus, the effect can solve the problem of glioblastoma cell resistance and prevent tumor recurrence by influencing cancer glioma stem cells.

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About the authors

V. A. Kolesnikova

Institute of Higher Nervous Activity and Neurophysiology of RAS

Author for correspondence.
Email: v.kolesnikova@ihna.ru
Russian Federation, Moscow

A. K. Mitina

Pirogov Russian National Research Medical University

Email: v.kolesnikova@ihna.ru
Russian Federation, Moscow

A. V. Ryabova

Natural Sciences Center of Prokhorov General Physics Institute RAS

Email: v.kolesnikova@ihna.ru
Russian Federation, Moscow

L. V. Fab

Institute of Higher Nervous Activity and Neurophysiology of RAS

Email: v.kolesnikova@ihna.ru
Russian Federation, Moscow

I. N. Pronin

Federal State Autonomous Institution «N. N. Burdenko National Medical Research Center of Neurosurgery» of the Ministry of Health of the Russian Federation

Email: v.kolesnikova@ihna.ru
Russian Federation, Moscow

G. V. Pavlova

Institute of Higher Nervous Activity and Neurophysiology of RAS; Federal State Autonomous Institution «N. N. Burdenko National Medical Research Center of Neurosurgery» of the Ministry of Health of the Russian Federation; Sechenov First Moscow State Medical University

Email: v.kolesnikova@ihna.ru
Russian Federation, Moscow; Moscow; Moscow

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2. Fig. 1. Expression of L1CAM in glioblastoma cell cultures and aptocytochemical staining of G01 L1CAM+ and G01 L1CAMcells with anti-L1CAM antibodies and labeled yly12-FAM aptamer. (а) – Levels of L1CAM expression in human glioblastoma cell cultures G01, Sus\fP2, Bl, Sh\fP3, Rozh. Data are represented as mean ± SD. n = 3 for each group. Statistically significant differences between the control and the treatment groups are indicated by asterisks (One-Way ANOVA, post-hoc Tukey HSD Test, ** – p < 0.01, *** – p < 0.001, **** – p < 0.0001). (б) – Aptocytochemical staining of human glioblastoma cells G01 L1CAM+ and G01 L1CAM- with yly12-FAM aptamer (green), and anti-L1CAM antibodies (red). Nuclei are stained with Hoechst 33342 (blue).

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3. Fig. 2. Results of the MTS assay for single and triple addition of aptamers to L1CAM to human glioblastoma cells. MTS-assays on the 10th day after exposure to L1CAM aptamers yly4, yly10, yly11, yly12, ylQ3 to glioblastoma cells (а) – G01, (б) – Sus\ fP2 in concentrations 10 mcM and 37.5 mcM. MTS-assays on the 10th day after exposure to L1CAM aptamers yly4, yly10, yly11, yly12, ylQ3 in concentration 10 mcM to glioblastoma cells G01 L1CAM+ and G01 L1CAM- (в) once and (г) every three days. Data are represented as mean ± SD. n = 3 for each group. Statistically significant differences between the control and the treatment groups are indicated by asterisks (Two-Way ANOVA, post-hoc Bonferroni Test, ** – p < 0.01, *** – p < 0.001, **** p < 0.0001).

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4. Fig. 3. Immunocytochemical staining of glioblastoma cells G01, Sus\fP2, G01 L1CAM⁺ and G01 L1CAM⁻ and PCR analysis of glioblastoma cells G01 L1CAM⁺ and G01 L1CAM⁻ after exposure to the aptamer to L1CAM yly12. (а) –Immunocytochemical staining of G01, Sus\fP2, G01 L1CAM⁺ and G01 L1CAM⁻ cells after exposure to yly12 aptamer on stem cell markers CD133, L1CAM, CD44, Nestin, Sox2 and proliferation marker ki67. Зел – green, кр – red. Nuclei are stained with Hoechst 33342 (blue). (б) – Real-time quantitative PCR associated with stemness CD133, L1CAM, CD44, Nestin, Sox2 and GFAP, in cell cultures G01 L1CAM⁺ and G01 L1CAM⁻ after exposure to yly12 aptamer. Statistically significant differences between the control and the treatment groups are indicated by asterisks (Two-Way ANOVA, post-hoc Bonferroni Test, * – p < 0.05, **** – p < 0.0001).

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