A 1,10-phenanthroline derivative selectively targeting telomeric G-quadruplex induces cytoprotective autophagy, causing apoptosis of gastric cancer cells

Aims: This study aimed to evaluate the ability of compound 13d to induce Autophagy and to promote Apoptosis of tumor cells and its interaction mechanism.

Materials and methods: Using CCK-8 assay, transwell assay, fluorescence resonance energy transfer melting analysis (FRET), transmission electron microscopy, flow cytometry assay, immunofluorescence assay, Western blot analysis, and wound healing assay.

Key findings: The results indicated that compound 13d could induce Autophagy and Apoptosis of gastric Cancer cells. Moreover, the findings of CCK-8 assay, colony formation, migration and invasion assay, and wound healing assay revealed that compound 13d would effectively inhibit cell proliferation, migration, and invasion. Its IC₅₀ value is about 2.4 μM against gastric Cancer cells, which is similar to positive drug‑platinum. 13d specific induction of telomere G-quadruplex formation was proved in extracellular FRET melting assay, and indirectly affected Telomerase activity. G-quadruplex formation promoted cell Apoptosis and Autophagy. Upon incorporating the Autophagy inhibitors 3-MA and HCQ, the expression of the Autophagy marker protein LC3 was then checked, suggesting that the compound 13d influences the Autophagy flux. Furthermore, knocking down the autophagy-related gene Atg5 to reduce the level of Autophagy enhances the anti-tumor activity and increases apoptotic cells' proportion. Mechanistic experiments have shown that blocking the Akt/m-TOR signal pathway plays a crucial role in Autophagy and G-quadruplex induced telomere dysfunction. DNA damage is the leading cause of Autophagy. Compound 13d combined with Autophagy Inhibitor can inhibit tumor cells more effectively.

Significance: Our findings demonstrate that compound 13d as a telomeric G-quadruplex ligand induces Telomere dysfunction, DNA damage response, Autophagy, and Apoptosis in gastric Cancer cells by blocking the Akt/m-TOR signaling pathway.