The Research Of P162Increase Radiosensitivity Of The Esophageal Cancer Cell Lines Ecal09by Inhibiting Chk1/2Expression

Objective：Cell cycle checkpoint kinase1(Chk1) and cell cycle checkpoint kinase2(Chk2) areimportant proteinkinases in the DNA danmage repair.new peptide P162have obviousanticancer effect and radiosensitivity to Eca109cell lines. Whether it increases theradiosensitivity of esophageal cancer cell lines Eca109by inhibiting the cell cycle checkpointsignal transfer pathway? We will Further understand the mechanism of radiosensitivity bychecking its inhibition of the expression of Chk1/2,and observe its influences on cell cycle;Method：1、Induce to form radioresistance cell lines Eca109R:Make esophageal cancer cell linesEca109as the research object，and give it small doses of repeated X-rays exposure to forma radioresistance cell lines Eca109R.AccumuLate exposure dose to60Gy，then make ithaving radioresistance in some distances;2、Check the growth inhibition rate of different doses (2、4、6、8Gy)of cell lines Eca109by MTT methods，and select the right dose;3、Detect the growth inhibition rate of different concentrations (5、10、20、40、60mg/L) ofP162of Eca109R cell lines by CCK-8method to determine the optimal drugconcentration needed for the experiment;4、Immunofluorescence test Chk1and Chk2positive expression in cell lines Eca109andEca09R；5、Divided the experiment into four groups: a group without P162and not expose to X-rays,a group without P162but expose to X-rays,a group with P162not expose to X-rays,agroup with P162expose to X-rays.Each group contains two cell lines Eca109andEca109R;6、Western blot detect the dynamic changes of Chk1and Chk2proteins expressed in the fourgroups;7、Measure the change of cell cycle with flow cytometry.ResuLt：１、Plank digestived cells whithout exposure to X-rays group and the group expose irradiation2,4,6,8Gy.Made them single cell suspension. The group without exposure as thecontrol,irradiation group as experimental group.Measure the OD value in24h、48h、72h,calcuLate the cell growth inhibition rate.The dose At half of the inhibition rate will bethe right dose for later experiment. Measure the cell growth inhibition rate at around50%in6Gy radiation dose，So take6Gy as the dose of radiation for follow-up experiments.�'、Select the optional P162concentration of Eca109R by CCK-8. Let5,10,20,40,60mg/L for the experimental group, the group without P162be the control group. Measure the ODvalue in24h、48h、72h, calcuLate the cell growth inhibition rate.About half of theinhibition rate will be the right concentration we choose. In20mg/L,the cell growthinhibition rate at around50%,so select20mg/L as the concentration of the test.Inaddition,We found in the experimental process， the cell growth inhibition rate willincrease as the concentration of P162increases.It means the inhibitory effect of P162were positively correlated to the concentration.３、Immunofluorescence showed:There is a small amount of Chk1and Chk2in the Esophageal cancer cell lines Eca109andEca109R.they express in the nucleus and cytoplasm, and the average fluorescenceintensity of Chk1and Chk2in Eca109R cell lines is higher than Eca109cell lines.itillustrates the expression of Chk1and Chk2in Eca109R exceed to Eca109.４、western blot resuLts display：①There are small amount of Chk1and Chk2in the esophageal cancer cell linesEca109.After6Gy irridiation,Chk1and Chk2show peak change as time changes,gradually increase,then gradually decline.②The group of esophageal cancer cell lines Eca109with P16248h still express Chk1andChk2.After6Gy irridiation,it Shows a trend of gradual decline over time.③Compare the group of esophageal cancer cell lines Eca109with P16248h and withoutP162,the Chk1and Chk2proteins drop in the group with P162. After irridiation for24hand48h, Chk1and Chk2proteins have reduced more pronounced in the group withP162；④There are certain amount of Chk1and Chk2express in Esophageal cancer cell linesEca109R.After irridiation,Chk1and Chk2gradual decline as time changes；At24h after6Gy irridiation，Chk1and Chk2are significantly decreased in the group with20mg/LP16248h.The group with40mg/L P16248h are decreased more significant，and less thanthe group of20mg/L P162；⑤The Chk1and Chk2expression in cell lines Eca109R higher than cell linesEca109,Compare the group with and without P162of Eca109and Eca109R,the expressionof Chk1and Chk2decline,Chk1and Chk2fall more apparently at24h afterirradiation.The Chk1and Chk2proteins significantly less in the group added40mg/LP162than with20mg/L of P162in cell lines Eca109R.In the Eca109and Eca109R celllines,The expression of Chk1exceed to Chk2，it reveals that Chk1may play a major rolein the formation of radiation resistance in esophageal Eca109. After6Gy irridiation, Chk1and Chk2expression are changing in the two cell lines with P162, but Chk1proteinchange more obvious, it illustrates P162may increase the radiation sensitivity of cell linesmainly by inhibiting the expression of Chk1protein.５、ResuLts of the cell cycle： ①Compared the cell lines Eca109with irradiation and without irradiation, G2phaseproportion significantly increased,it prompts G2/M phase arrest after the irradiation;②The G2phase proportion of the cell lines Eca109add20mg/L P16248h without or withirradiation less than the group without P162；③Compared the cell lines Eca109R without irradiation and with irradiation after24h，G2phase proportion increased,higher than the cell lines Eca109.It is clears that the arrest ofG2/M phase more obviously in the cell lines Eca109R, Eca109R have more radiationresistance;④Compare the group added20mg/L and added40mg/L P162of the cell lines Eca109R, G2phase proportion decreased,We can see the cell cycle block lifting more obvious alongwith the increase of concentration.Moreover, compare to the group without P162,G2phase proportion decreased;⑤Compare the group added20mg/L and added40mg/L P162after6Gy irridition，G2phase proportion decreased.It means the effect of P162is positively correlated withconcentration.Coclusion：1、Esophageal cancer cell line Eca109R have more radiation resistance than cell lines Eca109;2、The radiation resistance of esophageal cancer cell line Eca109R and Eca109associatewith the changes of Chk1and Chk2;3、P162can increase the radiosensitivity of esophageal cancer cell line Eca109R and Eca109;4、 P162increases the radiation sensitivity by inhibiting Chk1and Chk2proteinsexpression,removing cell cycle arrest, and taking the cells into the radiation relativelysensitive periods of M phase.