Effects Of A Fluorophore-Labeled Neomycin Conjugate On The Activity Of Mouse Breast Cancer 4T1 Cells

Based on the biological molecular targets for screening of antitumor drug lead compounds is one of research focuses in antitumor drugs.The G-quadruplex structure is a special secondary structure formed by the sequence of guanine-rich(G).The discovery of its relationship with the G-quadruplex structures and cancers by modern molecular biology techniques,as the antitumor drugs development provide a new opportunity.The compounds that can induce the formation of G-quadruplex structure or pecifically bind to G-quadruplex structure which can inhibit the growth of tumor cells,thus achieving the anti-cancer effect.Development of a G-quadruplex fluorescent probe with high selectivity for G-quadruplex,high stability of G-quadruplex and good biocompatibility will be the key to the detection of G-quadruplex structure,G-quadruplex distribution and functional studies provide important testing tools.This is of great significance to further study the interaction between small molecule ligands and G-quadruplex to develop targeted nucleic acid therapeutics.Neomycin is an aminoglycoside antibiotic.In recent years,researchers have found that neomycin can act as a small molecule ligand that binds to and stabilizes the G-quadruplex,which can combine with the groove of the G-quadruplex and stable G-quadruplex structure.Therefore,the present study will link the cyanine-based fluorescent dye Cy5.5 with neomycin via a lipophilic carbon chain in a conjugation manner.Design and synthesis of a novel fluorescently labeled neomycin molecular probe Neo-1 to study its effect on the activity of mouse breast cancer cells.To further investigate the mechanism of Nco-1 inhibiting mouse breast cancer 4T1 cells at the molecular level.A novel fluorescence-labeled neomycin molecular probe,Neo-1?was designed and synthesized by linking cyanine-based fluorescent dye Cy5.5 with neomycin via a lipophilic carbon chain in a conjugation manner.Using mouse breast cancer 4T1 cells as a model,MTS assay was used to examine the ability of the probe molecules to inhibit tumor cell proliferation.Using mouse breast cancer 4T1 cells as a model,the imaging capability of compound Neo-1 for tumor cells was examined by laser confocal fluorescence imaging,and the imaging region of compound Neo-1 tumor cells was performed using ribosome tracking reagents.Positioning research.To explore the mechanism of imaging and therapeutic effects of the probe compound Neo-1 on tumor cells at the molecular level.Using circular dichroism method and fluorescence polarization method,the binding ability and structural stability of the compounds Neo-1 and G-quadruplex were studied at the molecular level.Research shows that the new fluorescent compound Neo-1 has high fluorescence emission signal and good biocompatibility.The results of cell proliferation inhibition test showed that compound Neo-1 could inhibit the proliferation of mouse breast cancer 4T1 cells.With the increase of the concentration of compound Neo-1,the inhibitory ability was enhanced,and it was time-dependent,with an IC50 value of 12.0.?M.The effect of compound Neo-1 on the distribution of mouse breast cancer 4T1 cells detected by flow cytometry using PI staining method showed that the cells in G0/G1 phase decreased significantly with the increase of the concentration of compound Neo-1.<0.05),and decreased from 63.98%to 39.90%.In contrast,S phase cells increased significantly from 34.02%to 56.25%.Therefore,it was concluded that the compound Neo-1 can block 4T1 cells in the S phase,thereby inhibiting cell proliferation.The site of action of the compound Neo-1 in tumor cells was studied by laser confocal fluorescence imaging.The results showed that the compound Neo-1 disperses in the tumor cytoplasm and has high ribosome targeting ability.The interaction between the probe molecule Neo-1 and the two RNA G-quadruplexes(bcl-2 and TRF2 RNA G-quadruplex)formed in vitro was studied by fluorescence polarization and circular dichroism.As a result,the probe molecule Neo-1 was able to bind and stabilize the G-quadruplex structure.The compound Neo-1 acts on the bcl-2 RNA G-quadruplex ATm at 5?;the TRF2 RNA G-quadruplex ATm is 3.75?.Studies have shown that the new fluorescent compound Neo-1 has high fluorescence emission signal,G-quadruplex selectivity,high specific binding capacity and good biocompatibility,in the study of mouse breast cancer 4T1 cells as a model,The compound Neo-1 can achieve tumor diagnosis and treatment integration by binding and stabilizing the G-quadruplex structure.