DNA Damage Response And Growth Suppression Induced By MTH1 Inhibitors In Cancer Cells

Reactive Oxygen Species（ROS）is one of the most important molecules in life activities.However,the oxidation of intracellular biological macromolecules such as nucleic acids caused by excessive ROS can adversely affect cells’states.Free nucleotides especially deoxyguanosine triphosphate（dGTP）in the nucleotide pool are more susceptible to the oxidative damage than those on double-stranded DNA.The insertion of 8-oxo-dGTP into DNA can result in mispairing,mutations and cell death.MTH1（MutT homology 1）can prevent the damage caused by the high levels of ROS by sanitizing the oxidized dNTP such as 8-oxo-dGTP in the nucleotide pool efficiently.With the higher levels of ROS than normal cells caused by unusual proliferation and metabolism,tumor cells are much more dependent on MTH1,which drives the MTH1 to become a hot target for cancer treatment.Therefore,the development of novel and effective MTH1 inhibitors provides a new strategy for cancer therapy.In our previous study,we had synthesized and screened a series of potential new MTH1 inhibitors.We had shown that these small molecule compounds inhibit the activity of MTH1 strongly using the MTH1-catalyzed enzymatic reaction.However,whether these drugs can combine with intracellular MTH1 protein and inhibit its activity so that they can kill cancer cells deserves further study.Based on this,this article takes a deeper look at the influence of these underlying novel MTH1inhibitors on the activity of MTH1 and the growth of cancer cells.Firstly,the extent of interation between the compounds and the MTH1 proteins was detected using the Cellular Thermal Shift Assay（CETSA）.The results showed that the potential MTH1 inhibitors screened in previous experiments bind the MTH1protein tightly inside the cells.We next further explored the effect of C72-125 and 501513,binding with MTH1 protein strongly,on the activity of intracellular MTH1.We assume that if C72-125 and 501513 inhibit the activity of MTH1 in MCF-7 cells,the level of8-oxo-G,formed by the 8-oxo-dGTP accumulated in the nucleotide pool,will be increased.Immunofluorescence staining of 8-oxo-G showed that the level of8-oxo-G raised significantly in MCF-7 cells after treated with 14μM C72-125 or 1.6μM 501513 for 6 hours.The flow cytometry assay of 8-oxo-G got the same results.The 8-oxo-G in DNA can usually be identified and repaired by the Base Excision Repair（BER）pathway,causing DNA single-strand break or even DNA double-strand break.CASP image analyze of comet assay showed that the tail moment of MCF-7 cells increased dramatically after treated with C72-125 or501513 for 24 hours,supporting that the DNA breaks in MCF-7 cells is markedly increased.In addition,immunofluorescence staining of 53BP1,an important protein during the process of DNA double-strand break repair,showed that the ratio of53BP1 positive cells improved after drug treatment.It also revealed that C72-125and 501513 caused damage to the DNA in MCF-7 cells.We finally examined the effect of C72-125 and 501513 on the cancer cell growth.The MTT and colony-forming assay revealed that C72-125 and 501513inhibit the proliferation of MCF-7 cells in a concentration-dependent manner.The data detailedly indicated that IC₅₀ is 47.22μM after treated by C72-125 for 24 hours;IC₅₀ is 14.33μM after treated by C72-125 for 48 hours;IC₅₀ is 8.314μM after treated by C72-125 for 72 hours.The figures also showed that IC₅₀ is 13.06μM after treated by 501513 for 24 hours;IC₅₀ is 1.58μM after treated by 501513 for 48 hours;IC₅₀ is 1.239μM after treated by 501513 for 72 hours.Moreover,MTT assay also showed that the toxicity of C72-125 and 501513 to normal cells are lower than to MCF-7 cells.The flow cytometry results displayed that the cell cycle of MCF-7 cell was arrested in G₂/M phase after being treated with 7μM C72-125 for 24 hours and in G₁ phase after being treated with 1.6μM 501513 for 24 hours.Moreover,the rate of cell apoptosis increased remarkably after treatment,indicating that C72-125 and501513 can induced MCF-7 cell apoptosis.In summary,all the MTH1 inhibitors screened in previous experimental work bind with MTH1 tightly in MCF-7 cells.Among them,C72-125 and 501513 led to the level of 8-oxo-G increased markedly in MCF-7 cells.Moreover,C72-125 and501513 can induce DNA damage in MCF-7 cells,inhibit the proliferation of MCF-7cells,and change the cell cycle distribution and the percentage of apoptotic cells.The results of this study could provide scientific basis for cancer treatment by using these small molecule inhibitors of MTH1.