Combination Treatment Of Emodin And Telomerase Inhibitor Induces Significant Telomere Dysfunction And Cell Death

Objective:Telomere plays an important role in the stability of the genome.The high structure including G-quadruplex could induce telomere dysfunction,impede telomerase recognition,inhibit telomere elongation.It has been suggested that some small moleculars could bind and stabilize the structure,induce telomere damage,inhibit telomerase activity,then,inhibit the development of cancer.However,we observed that these telomere disorder could be restored by telomerase.Here we try to answer whether the combination of this G-quadruplex ligase and telomerase inhibitors can induced permanent DNA damage and inhibited tumor growth or not.Methods:(1)Here a structure-based virtual screeing assay was performed in a natural compound library to identify the novel G-quadruplex ligand.The stability of the ligand(emodin)for G-quadruplex was further demonstrated by the CD spectra and the UV melting.In addition,the effect of emodin on G-quadruplex in telomeric region was detected by IF-FISH in cells.(2)Cells were treated with emodin for different time,IF-FISH and FISH were used to detect the effect of emodin on genomic DNA damage,telomeric dysfunction.Cell apoptosis and senescence were detected by TUNEL and SA-β-gal assay.(3)Cells were treated with emodin for different time,telomere repeats amplification protocol and q-RT-PCR were used to detect the effect of emodin on telomerase activity.(4)Then the activity of telomerase was inhibited by telomerase inhibitors to detect telomerase positive cells,telomerase negative cells and telomerase-null normal cells proliferation and whether the above effects of emodin on cells would occur again.(5)To establish xenograft tumors mice model,combination treatment of emodin and telomerase inhibitor to observe the effect on tumor.Results:(1)Emodin was identified as one of the best candidates,which showed great binding potential to G4.Next,the emodin was confirmed to stabilize the formation of G4.(2)After 48 hours treatment of emodin,the frequency of genomic DNA damage,apoptotic and senescence cells were increased to the control.In addition,telomeric DNA damage and dysfunction increased with the emodin exposure to the control.The emodin induced TIFs and telomere abnormalities won’t last long and could be rescued with time.Furthermore,the number of apoptotic cells determined by TUNEL assay had the same trend.(3)TRAP assay revealed that the telomerase activity upon emodin exposure 48 hours was increased and mRNA expression of hTERT was also increased.(4)BIBR1532 worked synergistically with emodin to inhibited cell proliferation in telomerase positive cancer cells,however,in telomerase negative cancer cells and telomerase-null normal cells didn’t show additional inhibition.In addition,the cells were treated with emodin and telomerase inhibitor BIBR1532 for 10 days,it was observed that the recovery of telomere function was no longer observed.(5)In addition,the tumor growth of breast cancer mice treated with xenotransplantation was significantly inhibited by the combination of emodin and telomerase inhibitor BIBR1532.Conclusion:We identified the first time that,the emodin induced telomeric DNA damage could upregulate telomerase activity which may weaken its anticancer effect.And the combined usage of emodin and the telomerase inhibitor synergistically induced telomere dysfunction and inhibited and tumor generation.