Research On Role Of EGFR Family In Radioresistance Of Cancer Cells

Part 1Effects of HER2 inhibition on DNA-PK expression and activationin breast cancer cell line after irradiation[Objective] To observe effect of Herceptin on radiation induced DNA -pkcsexpression and its activiation in breast cancer cell lines, and elucidate the relationshipbetween herceptin and radiosensitization.[Methods] Survival fraction of breast cancer cell line treat or not treated withherciptin was observed after X-irradiation. DNA -pkcs expression was also investigatedusing Western blotting. Double strand break was detected by single cell electrophoreticcomet assay.[Results] Compared with control group, survival fraction of breast cancer cell linesafter radiation was significant decreased and expression of DNA-PKcs protein increased.after treated with herceptin survival fraction are further decreased with decreasedexpression of DNA-PKcs protein and activation However, Double strand break of breastcancer cell lines was further increased.[Conclusion] Herceptin raised breast cancer cell lines radiosensitization. Its possiblemechanism related to DNA-PKcs expression and activation inhibition and double strand break increasing.Part2AG825 increase radiosensitivity of breast cancer cell[Objective] To observe radiosensitive effect and proliferation inhibitive effect oftyrosine kinase inhibitor AG825 on breast cancer cell line with high expression of HER2 invitro. And to explore the mechanism by investigating effects of AG825 on DNA doublestand break and expression of DNA-PKcs protein.[Methods] MTT assay were used to observe the inhibitive effect of differentconcentrations (0,2, 5, 10, 20, 50, 100μM) of AG825 on proliferation of breast cancer cellline MDA-MB-453. To evaluate radiosensitivity effect of AG825, cells were divided into 3group: control group, group treated with radiation alone, group treated with radiation andAG825. Survival fractions after radiation of different groups were analyzed using cloneformation assay. To investigate possible mechanism of radiosensitive effect of AG825,DNA double strand breaks were determined by single cell neutral gel electrophoresis(comet assay) and western blotting was applied to detect expressions of DNA-PKcs protein.[Results]: MTT assay showed AG825 decreased proliferation rates of MDA-MB-453 cellsin a dose dependent manner. And survival fractions of cells irradiated alone weresignificantly lower than control group. Survival fractions of cells irradiated with presenceof AG825 were further decreased than cellls irradiated alone. In comet assay, tail momentof cel irradiated were higher than control group, and with presence of AG825 tail momentof cell irradiated further increased. However, western blotting showed, at different timepoints after radiation, expression of DNA-PKcs protein in cell irradiated with presence of AG825 was significantly lower compared with cells irradated alone.[Conclusion] AG825 could inhibit proliferation of breast cancer cell with highexpression of HER2. And AG825 could increase sensitivity of breast cancer cell lineMDA-MB-453 to radiation. The mechanism of its radiosensitivity effect may related toAG825's inhibition of radiation induced expression of DNA-PKcs protein which aremainly responsible for DNA double strand break repair.Part3Radiation induce HER2 nuclear transport in breast cancer cells[Objective] To observe the HER2 nuclear transport in breast cancer cell lines afterradiation and its possible role in radiation tolerance.[Methods] Confocal microscope and western blotting were applied to detect nuclearHER2 in MDA-MB-453 cells after radiation. And then cells were divided into 3 group:control group, group treated with radiation alone, group treated with radiation and drug.Effect of Herceptin, AG825 and Cisplatin on expression of nuclear HER2 were investigated.Survival fractions were also observed through comet assay. And immunoprecipitation wasapplied to detect complex of DNA-PK and HER2.[Results] After radiation, compared with control group, expression of nuclear HER2protein increased according to Confocal microscope and western blotting in breast cancercell with high expression of HER2 --MDA-MB-453. Cells treated with Herceptin, AG825showed, lower level radiation induced nuclear HER2 expression with decreased survivalfractions than control group. Cisplatin also induced nuclear HER2 expression in breastcancer cell with high HER2 expression. [Conclusion] Radiation could increase HER2 nuclear expression , and nuclearHER2 may correlate with radiation tolerance in breast cancer cells with high HER2expression.Part4Effect of radiation on EGFR nuclear translocationin Cervical cancer cell lines[Objective] To observe EGFR nuclcar translocation in cervical cancer cell linesafter radiation and its possible role in radiation tolerance of cervical carcinomas cell.[Methods] Expression of EGFR of 3 Cervical cancer cells lines were analysed. ThenWestern blotting were applied to detect nuclear EGFR and cytoplastic EGFR in Caski cellswhich showed hight level expression of EGFR after radiation. And cells were irradiatedwith or without Cetuximab, effect of Cetuximab on exprcssion of nuclear EGFR wereinvestigated. Survival fractions were also observed.[Results] Western blotting showed Caski cells have high level of expression of EGFR.And after radiation, compared with control group, expression of nuclear EGFR protein washigher in irradiated Caski cells than control according to western blotting with highexpression of EGFR. Cells treated with Cetuximab have lower level nuclear EGFRexpression with decreased Survival fractions than control.[Conclusion] Radiation could induce EGFR nuclear translocation in Cervicalcarcinoma cell lines and nuclear EGFR may correlate with radiation tolerance in Cervicalcarcinoma cell.