Effect Of Compound GA5 On Colorectal Cancer Cells' DNA Damage/Repair And Study On The Mechanism Of Sensitized Topo Inhibitors

Objective:To explore the effect and mechanism of compound GA5 on DNA topoisomerase(Topo)and the sensitizing effect of Topo ?nhibitorsMethods:In this study,human colorectal cancer cell HCT116 was used as a model:1.Single-cell gel electrophoresis was used to detect the effect of GA5 on DNA damage;2.TARDIS was used to detect whether GA5 stabilized Topo-DNA cleavage complex(Topec);3.qRT-PCR and Western blot to detect the effect of GA5 on HCT116 cells on the expression of Topo ? and DNA damage repair related factors(Chkl/2,PARP1,Tdp1);4.MTT method detect GA5 combination of TPT and vp-16 on the proliferation of HCT116 cells;5.Using mouse transplantation sarcoma S180 as a model,the tumor inhibition rate as an index(%),evaluate the inhibitory effect of GA5 combined with TPT and vp-16 on S180 growth;Thymus index and spleen index,liver coefficient and kidney coefficient as indicators,observe the toxicity of each administration group;comprehensively evaluate the effect of combined administration on tumor-bearing mice toxicity;6.Western blot to detect the effect of expression GA5 combination of TPT,vp-16 treatment HCT116 cells on Topo and Chkl/2,PARP1,Tdpl.Results:The results show that:1.GA5 has obvious comet-like tailing in short time and low or high concentration,which induces DNA double-strand break damage;2.GA5 stabilizes the Topo ? cc at higher concentration;No Topo ? acc was detected;3.GA5 down-regulated Topo ?,Chkl/2,PARP1 transcription in HCT116 cells at 24,48h,and down-regulated Tdp1 gene transcription at 8,24h;GA5 down-regulated Topo ?,Chkl,PARP1 protein expression levels in HCT116 cells at 24,48h,and down-regulated Chk2,8,24,and 48h Tdp1 protein expression level;although GA5 has different effects on the expression of various factors at different times,the overall expression of these factors is down-regulated;4.Compared with TPT and vp-16 alone,the combination of low-concentration GA5 and TPT and vp-16 has a significantly stronger inhibitory effect on the proliferation of HCT116 cells than the single use;5 GA5(1.25,2.5mg/kg)of tumor inhibition rate is 22.76,45.20%,TPT(0.2,0.3mg/kg)tumor inhibition rate was 30.34,59.02%,GA5(1.25mg/kg)+TPT(0.2mg/kg)tumor inhibition rate was 59.69%;vp-16(6.67,10mg/kg)tumor inhibition rates were 26.96,55.19%,GA5(1.25mg/kg)+vp-16(6.67mg/kg)55.22%.vp-16 can reduce the weight of mice when used alone,others have no significant effect on the weight of mice;TPT,vp-16 alone and combined with GA5 have inhibitory effects on the spleen and thymus of mice,which is equivalent to the tumor inhibition rate.Compared with single-use TPT and vp-16,the inhibitory effect was weaker;each administration group had no significant inhibitory effect on mouse kidney and liver.6.TPT alone can down-regulate the expression of Topo ? Chkl/Chk2,Tdp1 protein,up-regulate the expression of Topo ? a protein,GA5+TPT combination down-regulates the expression of Topo ?,Topo ? ?,Chkl/2,Tdp1 protein compared with TPT alone;vp-16 alone can down-regulate the expression of Chkl/2 and Tdp1 proteins,and up-regulate the expression of Topo ? a protein.GA5+vp-1 6 combined with vp-16 alone can down-regulate the expression of Topo ?,Topo ? ?,Chk1/2,and Tdp1 proteins.the expression of PARP1 at different times in each group has no significant effect;Low concentration of GA5 can up-regulate the expression of Topo ? a protein,and has no significant effect on the expression of other proteins.Conclusions:The compound GA5 stabilizes the Top I cc,is a Topo ? poison,and simultaneously inhibits the expression of Topo ?.The mechanism of action on Topo ? has not yet been determined.GA5 can also directly act on DNA to cause DNA breakage damage;it also inhibits expression of DNA damage repair related factors.The compound GA5 combined with TPT and vp-16 can increase the sensitivity of HCT116 cells to Topo inhibitors in vitro,and can increase the sensitivity of mouse transplanted sarcoma SI 80 to chemotherapeutic drugs in vivo,and the toxicity is relatively reduced,showing a synergistic effect.Due to the complex effects on various factors,its sensitization mechanism is still unclear.