Interaction analysis of anti-cancer drug Methotrexate with bcl-2 promoter stabilization and its transcription regulation

Abstract

G-quadruplex (G4) motifs are higher-order secondary structures putatively present in the promoter region of several oncogenes and telomeric ends of chromosomes. The stabilization of G-quadruplex structures in the promoters of proto-oncogenes using specific small molecules provides a prominent strategy for the development of anti-cancer therapeutics. Methotrexate is an FDA-approved anti-cancer drug that is known to inhibit dihydrofolate reductase (DHFR) activity that leads to cell death. Here, in this study, we employed a broad range of bio-physical techniques to understand the interaction of Methotrexate with G-quadruplex motifs present in the proto-oncogenes (bcl-2, c-myc, c-kit21) and telomeric region (tel22). Interestingly, Methotrexate showed the highest binding affinity and specificity for bcl-2 G4 (Kd1 = 9 nM) as compared to other G-quadruplex, and duplex DNA. Circular dichroism melting analysis depicted higher stabilization of bcl-2 G4 by the Methotrexate. While molecular docking and dynamic simulation analysis revealed stacking mode of interaction with the G4 structure. Furthermore, in a cellular-based assay, Methotrexate demonstrated higher toxicity against A549 lung cancer cells over other cancer cells such as PC-3, HeLa, and HepG2. Quantitative RT-PCR and western blot results provide direct evidence that Methotrexate treatment to A549 lung cancer cells could downregulate the transcription and translation of the bcl-2 with the stabilization of G-quadruplex motif. In summary, this report provides valuable information about the alternative molecular mechanism of Methotrexate for the treatment of cancer and offers new insight into its anti-cancer activity.