P162Enhance The Radiosenstivity Of Esophageal Carcinoma Cell Eca109by Inhibiting The Transcription Factor Of Hedgehog Signal Gli-1

AIM: To investigate whether the novel peptide P162,targeting G3BP（RasGAP bindingprotein）,enhance radiosensitivity of esophageal carcinoma cell line Eca109,characterized by inhibiting the transcription factor of Hedgehog signal Gli-1or not．METHODS:1.Repeatedly irradiate (a total irradiation dose of60Gy) Eca109to induceesophageal carcinoma cells Eca109R.2. Eca109and Eca109R cells was irradiated at0、2、4、6、8Gy,whoseinhibition of cell proliferation were determined by Cell Counting Kit assay, thendistingishing both cells.3. The expression of Gli-1in Eca109and Eca109R cells was detected byimmunohistochemistry and immunofluorescence.4. Eca109and Eca109R cells were treated by20μmol/L P162.Then after48hours, HE staining was employed to observe their morphology changes.5. After irradiated in1,2,4,24,48,72h,7,10,14and18d point-in-time,Eca109cells, was detected by Western blot in order to determine the expression of Gli-1in nucleus, compared to nonirradiated control cells. Western blot was employed todetermine the expression of Gli-1in nuclear Eca109R cells, which were irradiated after48h, compared to nonirradiated ones.6. The expression of Gli-1in disposed group cells were detected byWestern blot. Firstly the disposed group cells were treated by20μmol/L P162in48h,then irradiated by6Gy, including the following four groups: non-irradiation withmedicine group, irradiation with medicine group, non-irradiation without medicine group,irradiation without medicine group. Each group contains Eca109, Eca109R.7. Apoptosis in disposed group cells was determined by flow cytometry.RESULTS:1. Eca109R possessing certain radiation resistance displayed lower ability ofgrowth inhibition than Eca109in the same irradiation dose and time.2. In terms of immunohistochemistry and immunofluorescence result,the Gli-1protien were expressed in cytoplasm and nucleus in Eca109and Eca109R cells[1.05629±0.098vs1.703975±0.055(P＜0.05),39.9567±0.0097%vs66.4589±0.0251%(P＜0.05)]. Eca109expressed lower Gli-1than Eca109R.3. HE staining result：undisposed Eca109cells were round and anomalouspolygon,such as paving stone. Untreaded Eca109R cells were irregular type spindle andanomalous arrangement.While, Eca109cells, disposed by20μmol/L P162after48h,were shrinking and aggregated with nucleus splitting and mixing. Eca109R cells werealso buckling and merged,with nucleus cripping, splitting and blending.4, The result in the expression of Gli-1in nucleus after irradiated in1,2,4,24,48,72h,7,10,14and18d point-in-time were as follows: compared withnonirradiated control Eca109cells, firstly, the expression of Gli-1in nucleus weredescread, minimum at48h time point, afterwards increased, maximum at14d timepoint.Lastly,it were inclined to decline. Compared with nonirradiated Eca109R cells, theexpression of Gli-1in nuclear Eca109R, which were irradiated after48h, were reduced.5. The result in the expression of Gli-1in nuclear Eca109andEca109R afterirradiated and P162treated were as follows: non-irradiation with medicine groupexpressed lower Gli-1protein than non-irradiation without medicine group. Innon-irradiation without medicine group, Eca109expressed lower Gli-1than Eca109R(F=135.73, P<0.0001); Irradiation with medicine group(after irradiated2d,14d)expressed lower Gli-1protein than irradiation without medicine group (P＜0.05), Inirradiation without medicine group, the expression of Gli-1in nuclear Eca109andEca109R has no statistical difference [respectively,F=1.89, P=0.2064(2d),F=0.45,P=0.5191(14d)].6.P162combined radiotherapy facilitated cells apoptosis.CONCLUSION: GLI-1in nucleus was relevant with radioresistance of esophagealcancer. The radiosensitization effect on Eca109of the novel peptide P162was associatedwith inhibiting the transcription factor Gli-1and promoting apoptosis．...