Marein Inhibits Poly(ADP-ribose) Polymerase 1(PARP1) And Enhance Radiation Sensitivity Of Colorectal Cancer Cells

Objective:To investigate the role of Marine as A Poly(ADP-ribose)Polymerase-1(PARP1)inhibitor in enhancing the irradiation sensitivity of HCT116(Human colon cancer cell)and MC38(mouse colon cancer cell).Methods:(1)For cell proliferation assays,HCT116 and MC38 were pretreated by different concentrations of Marein(5,10,20 ??)for 24 h before irradiated(6Gy),then cell number was calculated using the MTT assay.HCT116 and MC38 were pretreated by Marein 10?? for 24 h before irradiated(2Gy,4Gy,6Gy,8Gy),Finally,cell number was calculated using the MTT assay.(2)In clonogenic assays,HCT116 and MC38 were pretreated by Marein10?? for 24 h before irradiated(6Gy).Finally,colonies were scored.(3)HCT116 and MC38 were pretreated by Marein10?? for 24 h before irradiated(6Gy),Western Blot was used to evaluated the formation of Poly ADP-ribose(PAR)polymers chains,the capability of PARP1 trapping by Marein,the protein levels of ?-H2 AX and Cleaved-PARP.(4)HCT116 cells were pretreated by Marein10?? or Olaparib10?? for 24 h before irradiated(6Gy),?-H2 AX focus-positive HCT116 cells was were counted by immunofluorescence.Results:(1)MC38 cells were pretreated by different concentrations of Marein(5,10,20??)for 24 h before irradiated(6Gy).MTT assays demonstrated that the proliferation capacity of MC38 showed decreased in Marein concentration dependence(P<0.01).As HCT116 cells pretreated by lower concentration of Marein(5??),the proliferation capacity decreased was not statistical significance(P>0.05),but was significantly decreased by moderate or higher concentration of Marein(10,20??)(P<0.01).HCT116 and MC38 were pretreated by Marein10?? for 24 h before exposed to different radiation doses(2Gy,4Gy,6Gy,8Gy 6Gy),Finally,Their ability of proliferation was also significantly reduced(P<0.05).(2)Colony formation assays show that HCT116 and MC38 cells were all markedly sensitivity to Marein 10?? after exposed to radiation(6Gy)(P< 0.05).(3)In the blank control group,the PAR chain level was relatively low in HCT116 or MC38 cells.Interestingly,after X-ray 6Gy irradiation,the formation of PAR chain was obviously induced and Marein 10?? could significantly inhibit the formation of PAR chain.From the perspective of time gradient(15min,30 min,45min after irradiation),the time of PAR chain formation induced by irradiation and reduced by Marein in the two cells were differently.After irradiated with colorectal cancer cells HCT116 and MC38,the Marein significantly drived trapping of PARP1 around the chromatin in a relatively short period of time.Marein significantly increased the expression of ?-H2 AX and the production of Cleaved-PARP induced by irradiation.(4)Immunofluorescence experiments showed that both 10 ?M of Marein and 10 ?M of Olaparib significantly increased the number of ?-H2 AX nuclear positive HCT116 cells induced by irradiation.Conclusions:(1)The Marein in the traditional Chinese medicine Xueju is a potential PARP1 inhibitor.(2)The colorectal cancer cells PARP1 inhibition by Marein can reduced the formation of its auto-PARylation product PAR chain,and trapped PARP1 to form PARP1-DNA complexes around chromatin.(3)Marein has no anticancer effect,but as a PARP1 inhibitor,can significantly enhances the radiation sensitivity of colorectal cancer cells,they can exert radiation sensitization both in human colorectal cancer cells and mouse colocrectal cancer cells.