Study On The Mechanism Of Oxocrebanine On DNA Damage In MCF-7 Cells Based On Topoisomerase Inhibition

Oxocrebanine is an aporphine alkaloid monomer compound extracted from the traditional Chinese medicine Stephania Hainannensis H.S.Lo et Y.Tsoong.Our previous data showed that oxocrebanine had certain anti-tumor activity in vitro experiments,and the sensitive cell line was MCF-7(IC₅₀value was 16.66 mol/L),oxocrebanine could induce autophagy in breast cancer MCF-7 cells.Some aporphine alkaloids interfered with cell cycle and inhibited cell growth through the inhibition of topoisomerase.Topoisomerase plays an important role in DNA replication and transcription.In order to further explore the causes of oxocrebanine cytotoxicity on human breast cancer MCF-7 cells,this study started with topoisomerase for further research.To observe whether the oxocrebanine caused DNA damage in human breast cancer MCF-7 cells.The inhibition of oxocrebanine on topoisomerase was investigated by DNA unwinding assays and k DNA untying assays.DNA unwinding assays shown that the 100μmol/L oxocrebanine can completely inhibit the activity of 1 U topoisomeraseⅠ,0.5μg p BR322DNA remained superhelix state,and the oxocrebanine of 25μmol/L had basically no inhibition to 1 U topoisomeraseⅠ,all the superhelix DNA were unwinding in the test system.The oxocrebanine of 25μmol/L can completely inhibit 1 U topoisomeraseⅡαactivity,that was different from the oxocrebanine to topoisomeraseⅠinhibition degree.KDNA melting assays was the specificity experiments to evaluate topoisomeraseⅡactive.That results was the same as the DNA unwinding assays,low dose（25μmol/L）oxocrebanine can completely inhibit 1 U topoisomeraseⅡαactivity,which further proved the inhibitory activity of oxocrebanine to topoisomeraseⅡα.oxocrebanine can not only inhibit the function of topoisomeraseⅡαrelaxation DNA,but also inhibit that the topoisomeraseⅡαmediated DNA rupture.The experimental results show that oxocrebanine was the dual inhibitor of topoisomeraseⅠand topoisomeraseⅡα.DNA breaking assays and DNA embedding assays were used to explore the detailed mechanism of inhibiting topoisomerase by oxocrebanine.No linear DNA was formed in DNA breaking assays,so it was known that the oxocrebanine was a catalytic inhibitor.DNA embedding assays revealed that 100μmol/L oxocrebanine could embed DNA to make the superhelix p BR322 DNA still coiled.The experimental results showed that oxocrebanine was a catalytic inhibitor of topoisomerase and a DNA embedding agent,and its ability of embed DNA was increased with the oxocrebanine concentration.TopoⅠ、TopoⅡα、γH2AX、ATM、p-ATM、ATR、p-ATR、CHK1、p-CHK1、CHK2、p-CHK2、p-H3 proteins were detected by Western blotting.The experimental results indicated that oxocrebanine could reduce TopoⅠand TopoⅡαprotein expression as a dose dependent.This suggests that the oxocrebanine can degrade TopoⅠand TopoⅡαprotein.γH2AX was a DNA damage marker protein.The increase ofγH2AX shows oxocrebanine was causing DNA damage in MCF-7 cells.Interestingly,oxocrebanine doesn’t activate ATM-CHK1/CHK2 pathway like etoposide,which was the typical DNA damage repair stress.The site of ATR Ser428 phosphorylation was increased.The total protein of CHK1 was decreased,but the expression of its Ser286 phosphorylation sites protein was aggrandized.CHK2 total protein has no obvious change,phosphorylation levels of CHK2 Thr68 was decreased.P-H3was a marker protein of the M phase in the cell cycle,because the expression of histone H3only occurs in the M phase,and the increase of phosphorylated H3 means that cell cycle changes may occur.In conclusion,oxocrebanine was a dual inhibitor of topoisomerase I and topoisomerase IIα.Oxocrebanine can be embedded in DNA,and it was a catalytic inhibitor of topoisomerase.Oxocrebanine can down-regulate TopoⅠand TopoⅡαproteins.DNA damaged when treated with oxocrebanine in MCF-7 cell,and the ATR-CHK1 pathway which was related to DNA damage repair was activated.the phosphorylation of CHK2 protein was inhibited.The phosphorylation of histone H3 was increased.By inhibiting topoisomerase,oxocrebanine can further cause DNA damage in human breast cancer MCF-7 cells,thereby affecting the proliferation of MCF-7 cells.