The Radiosensitization Effects Of SRC-1 In Glioblastoma

Background : Glioblastoma multiform(GBM)is the most malignant glioma of astrocytomas which accounts for about 54% of all gliomas.Clinically for glioblastoma treatment is surgery adjuvant radiotherapy and chemotherapy.As GBM is a highly aggressive tumor and most in the important structure of the brain,the current technique of surgery is difficult to achieve complete resection,so it is easy to relapse and has higher invasive.Because of the heterogeneity of tumor cells,intracranial pressure and blood brain barrier,most of the chemotherapy drugs have limited effects.The mechanisms of radiotherapy resistance,tumor invasion and lack of oxygen in prognosis are still unclear.The relapse after treatment is not only related to the tumor cells themselves,also related to the tumor microenvironment of radiotherapy resistance.Radiotherapy sensitization of glioma involved multiple signaling pathways.The steroid receptor assisted activating factor-1(SRC-1,also known as NCOA-1)is a member of the steroid receptor-assisted activator(SRC)family.SRC-1 has two homologous proteins SRC-2 and SRC-3.SRC-1 m RNA is widely expressed in the brain,lung,heart,liver,kidney,stomach,thyroid and other organs and tissues.SRC-1 is related in many important physiological activities such as the development and maturation of the brain,the glycogenogenesis in the liver and the oxidative metabolism of the steroids,the reproductive function of uterus and prostate,the formation of the breast.At the same time SRC-1 is also associated with a variety of diseases,it may be involved in such as cancer,diabetes,obesity,high blood pressure and central nervous system degenerative diseases.In the field of cancer,SRC-1 research focused on breast cancer.SRC-1 has almost no expression in normal mammary epithelial cells,but high expression in 20% of breast cancer.In vitro studies have shown that SRC-1 can activate AP-1,HOXC11,Ets-2 and other auxiliary activating factors,and further up-regulate the expression of transcription factors twist 1,c-Myc,S100 calcium adduct protein B to promote breast cancer proliferation and invasive.Studies have shown that SRC-1 and SRC-3 activated androgen receptor,estrogen receptor α and β to involved in tumorigenesis and progression in astrocytomas.The effect of SRC-1 on glioma radiotherapy has not been reported.In this study,we aimed at the radiosensitization effects of SRC-1 in human astrocytoma cell line U251.Methods:(1)Use lentivirus infection to construct stable knock down SRC-1 U251 cell line.(2)The effect of knockdown SRC-1 on radiotherapy of U251 cells line was detected by irradiation and clone formation assay,then the cell survival curve was plot.(3)The expression of γ-H2 AX in knockdown SRC-1 U251 cell line was measured by immunofluorescence after irradiation.(4)The effect of knockdown SRC-1 on DNA damage repair in U251 cells was detected by comet assay.(5)The expression of related proteins in DNA damage repair pathway was detected by Western Blot assay,and the mechanism was analyzed.Results:(1)Through the clone formation experiment,irradiation shows significantly proliferation inhibition on SRC-1 knockdown U251 cells.(2)Immunofluorescence shows the high expression of γ-H2 AX in knockdown SRC-1 U251 cells,it demonstrating that the DNA damage caused by radiation is significantly enhanced.(3)The comet assay detected knockdown SRC-1 U251 cells still have trail after 24 hours irradiation,this indicated that knocking down SRC-1 reduced the ability of DNA damage repair in U251 cells.(4)Western Blot showed that the total ATM protein expression decreased after SRC-1 been knock down,and the expression levels of phosphorylated ATM,Chk2 and Chk1 were inhibited after irradiation.Conclusions:In this study,After SRC-1knocked down in human glioblastoma cells U251,we use clonal formation,immunofluorescence,and comet assay to detect the effects of SRC-1 in cell proliferation,expression of γ-H2 AX and the ability of DNA damage repair after irradiation.Then detected DNA damage repair related proteins by Western Blot.We proved SRC-1 plays an important role in the radiotherapy of U251 cell.Knocking down SRC-1 can inhibit DNA damage repair ability in U251 cells and enhance its radiosensitization.