TUMOR NECROSIS FACTOR-?? REPRESSES ANDROGEN SENSITIVITY IN THE LNCaP PROSTATE CANCER CELL LINE

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Abstract
Prostate tumor progression is characterized by development of androgen independence and a heterogeneous distribution of the androgen receptor (AR). Tumor necrosis factor alpha (TNFalpha) has been demonstrated to contribute to the progression of several cancers and thus may play a role in prostate cancer progression. Accordingly, we examined if prostate cancers express TNFalpha and the effect of TNFalpha on androgen sensitivity and AR expression in LNCaP prostate cancer cells. Immunohistochemical analysis of prostate tissues, ELISA, and northern blotting of LNCaP cell lines were carried out for detection of tumor necrosis factor-alpha (TNFalpha). To see the effect of TNFalpha on androgen receptor (AR), western blotting and northern blotting were performed after extraction of total protein and total RNA from LNCaP cells. Regulation of androgen-sensitivity by TNFalpha was investigated with cell proliferation assay and luciferase assay using PSA promoter after transfection of LNCaP cells. Immunohistochemical analysis demonstrated that TNFalpha protein was strongly expressed in epithelial cells of prostate cancer tissue but not in normal prostatic tissue. Basal level of TNFalpha in cell culture medium from LNCaP cells was very low. However, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced TNFalpha secretion into medium up to 1600 pg/ml/day. Furthermore, 24 hr. post-TPA treatment TNFalpha mRNA levels were increased 15-fold compared to pre-treatment levels. TNFalpha (0 to 30 ng./ml. for 4 days) repressed AR protein and mRNA levels in a dose-dependent fashion in LNCaP cells. Pre-treatment of cells with actinomycin D treatment revealed that repression of mRNA levels was exerted at the post-transcriptional level. TNFalpha inhibited the ability of 10-9 M dihydrotestosterone (DHT) to induce LNCaP cell proliferation and activation of the prostate specific antigen (PSA) gene promoter. This inhibition was partially reversed by overexpression of transgenic androgen receptor. TNFalpha is present and inducible in prostate cancer cells and short-term TNFalpha diminishes androgen-sensitivity in LNCaP cells through down-regulation of AR protein and mRNA levels. These results suggest that TNFalpha may play a role in the initiation of an androgen-independent state in prostate cancer through its ability to inhibit AR sensitivity in prostate cancer.