The Bazhen decoction reduced the presence of G4 structure and endowed the cellular resistance to DNA replication stress induced by G4 stabilizer
We further investigate the impact of Bazhen decoction on G4 structure, DNA replication stress induced DNA damage, cell cycle progression. We first used anti-G4 antibody to detect the amount of G4 structure in G3DKO cells before and after Bazhen decoction treatment. We found that the signals for G4 structure were obviously diminished after Bazhen decoction treatment (Figure 3, A, B, G4), and the signals for DNA helicase Recql4 (Figure 3, A, Recql4) and Blm (Figure 3, B, Blm) were enhanced, and colocalized with G4 structure. These data suggest that the Bazhen decoction promote the resolving of G4 structure by facilitating the recruitment of DNA helicases Recql4 and Blm to G4 DNA. To further verify these data, we applied the G4 stabilizer pyridostatin (PDS) to stabilize G4 structure and induce DNA replication stress (DNA damage). As expected, comparing with control cells, the PDS treatment stabilized and increased the amount of G4 structure detected, while the Bazhen decoction could obviously diminish the G4 signals stabilized by PDS (Figure 3, A, B, G4).
We also revealed that the PDS treatment could down-regulate the DNA helicase Blm, Recql4, Parp1, Mcm7, telomere protein Terf1, and cell cycle protein Cdc2, Cdk2 in G3DKO cells, while Bazhen decoction could rescue the expression of these proteins (Figure 4, A). The cell cycle analysis revealed that Bazhen decoction could promote the cell cycle progression, reduced the percentage of G1 phase cells, and increase the percentage of S phase and G2 phase (Figure 4, B). The PDS treatment also induced the increase of G2 cells, which might due to the DNA damage repairing events induced by DNA replication stress. The Bazhen decoction application further increased the G2 phase cells, which might ensure the proper repair of DNA damage induced by PDS. To verify this, we detect the DNA damages induced by PDS with/without Bazhen decoction. We could observe the signal of DNA damage marker γ-H2AX in the G3DKO cells without any treatment (Figure 4, C, control), this might due to the background DNA replication stress induced by Wrn and telomerase knock out. The application of Bazhen decoction obviously reduced theγ-H2AX signal, indicates that the DNA damages induced in premature aging cells were repaired (Figure 4, C, BZT). Compared to these, the cells treated with PDS showed increasedγ-H2AX signals, indicated the induction of DNA damages by PDS (Figure 4, C, PDS). While the application of Bazhen decoction obviously diminished theγ-H2AX signals induced by PDS, indicates that Bazhen decoction could facilitate the repair of DNA damages induced by G4 stabilization (DNA replication stress) (Figure 4, C, PDS+BZT).
Together these data suggest that the Bazhen decoction could promote the resolving of G4 structure and endow the cellular resistance to PDS induced DNA replication stress, thus enhance the quality control of cell cycle.