The Study Of Arsenic Trioxide’s Effect On Keap1Gene Inprostate Cancer Cells DU145

Background and PurposeProstate cancer is the most common malignancy in the male genitourinary system.Currently the main method of surgical treatment of prostate cancer, hormone antagonist therapy, chemotherapy and radiotherapy.Although the majority of patients with prostate cancer androgen deprivation initially effective, but they are usually a few months to a few years later appeared in androgen-independent. Prognosis androgen-independent prostate cancer is relatively poor, and many of the chemotherapy and radiation therapy is not sensitive. Currently, The mechanism of hormone-independent prostate cancer resistant to chemotherapy and radiation therapy is unclear. Therefore, The mechanisms of tumor cells reverse drug and how to reverse drug resistance are important issues need to be resolved.In recent years, a number of trials have confirmed that KEAP1-NRF2-ARE signaling pathway associated with multiple resistance mechanisms [1,2],The current KEAP1gene mutations have been found in the NSCL, breast cancer, colon cancer and other tumor tissues, So NRF2-related protein expression associated with decreased expression of the protein [3,4], has been confirmed by experiments in non-small cell lung cancer, colon cancer, prostate cancer DU145cells KEAP1gene promoter CpG island hypermethylation and low expression [5,6],PC3prostate cancer cells with high expression of the gene KEAP1,So the PC3cells was chosen as a positive control group [7].Chinese scholars use arsenic trioxide for acute promyelocytic leukemia achieved a significant effect, caused widespread concern; subsequently confirmed its solid tumors can get the same effect, while demethylation of arsenic trioxide have been reported. In this study, different concentrations of As2O3solution DU145cells after treatment for prostate cancer detected on cell proliferation and inhibition of KEAP1affected cells, NRF2protein expression on preliminary exploration KEAP1gene As2O3on possible mechanisms, so as androgen-independent prostate cancer treatment provide new therapeutic drugs.Materials and methods1. The androgen-independent prostate cancer DU145cells were routinely cultured with DMEM-H culture medium (containing10%fetal bovine serum) in a cell incubator (temperature:37°C,5%CO2); PC-3cells were routinely cultured with RPMI-1640culture medium (containing10%fetal bovine serum) in a cell incubator (temperature:37°C,5%CO2);2．MTT assay was used to test the growth of DU145cells after the treatment of As2O3at different concentrations (0、0.5、1.0、2.0、4.0、6.0、8.0μmol/L) for24,48,72h，and to draw the curve of growth inhibition.3．RT-PCR was used to detect the mRNA expression of KEAP1genes in DU145cells,Which treated with different concentrations (0、0.5、1.0、2.0、4.0μmol/L) As2O3after72h;4．Western blot was used to detect the protein expression of KEAP1genes in DU145cells,Which treated with different concentrations (0、0.5、1.0、2.0、4.0μmol/L) As2O3after72h, PC3cells group as a control group;5．Statistical analysis: Experimental data was processed by SPSS17.0statistical software, Statistical differences were compared using non-parametric tests， t-test and analysis of variance, inspection level α=0.05. Results1. MTT results showed that: With As203concentration increased inhibition of DU145cells were more obvious, the highest inhibition rate at8.0μmol/L; based on the measurement of the inhibition rate at different time points, the highest inhibition rate at the time of72h. So As203on DU145cells in vitro in a dose-dependent and time-dependent manner;2. RT-PCR results showed that: The mRNA of KEAP1gene of group which not treated with As203expressing no obvious, after treated with As20372h, The mRNA of KEAP1gene expression gradually increased, At2.0μmol/L, the bands has the maximum brightness, The KEAP1gene has the strongest expression;3. Western blot showed that: In negative control group, keap1protein is notexpressed; After the human prostate carcinoma DU145cells treated with different concentrations of As2O3by72H, Protein of KEAP1were expression appeared in each concentrations groups expression appeared.The KEAP1protein bands are deepest in treatment group of2.0μmol/L and the positive control group display of PC-3.ConclusionsWithin a certain range of concentrations of arsenic trioxide. The prostate cancer DU145cells inhibited proliferation, and its inhibition increased with dose and time dependent manner, In a certain range of concentrations of arsenic trioxide can induce the re-expression of KEAP1mRNA and protein.