P04.19 A combined use of a G-quadruplex oligonucleotide (GQ) and neuro-inducing small molecules inhibit glioblastoma cell proliferation
Authors: Kolesnikova V , Samoylenkova N , Drozd S , Revishchin A , Usachev D Y , Kopylov A , Pavlova G 
Published: September 2021

Abstract

BACKGROUND
According to one of the theories, gliomas can occur as a result of dysregulation of stem cell division in the subventricular region of the brain. The CD133 membrane marker is a characteristic of both normal and tumor neural stem cells therefore it can be used to isolate a stem cell population from tumor tissue. Tumor cells actively proliferate which suggests that their possible differentiation may be achieved by inhibiting of their division as these two processes are mutually exclusive. For this purpose, G-quadruplex oligonucleotides together with neural-inducers such as a brain-derived neurotrophic factor (BDNF) may be used.

MATERIAL AND METHODS
Five cell cultures obtained from human glioblastoma tissues were analyzed for expression of CD133 using RT-qPCR. From cell culture with the highest level of CD133 using immunomagnetic separation CD133+ and CD133- cultures were received. CD133fr/peGFP-c1 recombinant DNA consisted of a CD133 second extracellular loop fragment and a peGFP-c1 vector was constructed to determine the localization of prominin-1, that is known as CD133 when found on cell membrane, using confocal microscopy. On chosen cell cultures an oligonucleotide bi-(AID-1-T) and its combination with BDNF were tested. The mechanism of GQ’s action is cytostatic and its non-toxicity properties were proved by flow cytometry. For evaluating the proliferative activity of cells MTT assay was performed on 10th and 20th days after exposure to the factors.

RESULTS
Cell culture G01 was chosen for further research as it had the highest level of the CD133. Colocalization of CD133 and GFP demonstrated a membrane localization of CD133 in cells with high expression level of this marker. MTT assay on 10th day after exposure to bi-(AID-1-T) as well as its combination with BDNF on cell culture G01 CD133- showed total inhibition of cell proliferation. The same combinations tested on G01 CD133+ cell culture demonstrated no difference in proliferative activity. After 20 days after exposure to bi-(AID-1-T) and combination of bi-(AID-1-T) with BDNF the significant decrease of G01 CD133+ cells’ proliferation was observed. When tested on whole glioblastoma cell culture G01 these combinations also showed significant inhibition of cell proliferation.

CONCLUSION
We showed that glioblastoma cells upon transfection with recombinant DNA, that contains a fragment of CD133, mainly have a membrane localization of this marker. It was observed that CD133+ cells are more stable to external influence that can be a proof of the fact that CD133 is charactered for glioblastoma stem cells. We tested the effect of an GQ bi-(AID-1-T) and its combination with BDNF and showed that BDNF is necessary for blocking proliferation of glioblastoma cells. Altogether, the results may be used for further research as it reveals a potential treatment for patients with glioblastoma.
Grant №075-15-2020-809 (13.1902.21.0030).