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.