Role of androgen receptor in CK5 +CK8 + cells isolated from prostate cancer LNCaP cells and its regulating mechanism / 肿瘤研究与临床

ABSTRACT

Objective:To investigate the role of androgen receptor (AR) in CK5 +CK8 + cells isolated from prostate cancer LNCaP cells and its regulating mechanism. Methods:CK5 +CK8 + cells were isolated from LNCaP cells by using flow cytometry. Lentivirus vector carrying AR gene was transferred in CK5 +CK8 + cells. The experiments were divided into AR CK5 +CK8 + group transfering AR and V CK5 +CK8 + group transfering blank load. The expressions of AR, p-AKT and bcl-2 were tested by using Western blot assay under different concentrations of androgen (1 nmol/L and 10 nmol/L dihydrotestosterone). Methyl thiazolyl tetrazolium (MTT) assay, cell migration assay and soft agarose gel clone formation assay was used to detect the effect of AR on the biological property of CK5 +CK8 + cells. The effect of activated inhibitors such as LY 294002 (LY), γ-tocotrienol (γ-TT) and/or 5-fluorocytosine inducing AR expression (5-AZA) through AKT signal pathways on CK5 +CK8 + cells proliferation was detected by using MTT assay. Results:After AR gene was transferred into CK5 +CK8 + cells, the expression of AR was increased, while the expression of p-AKT and bcl-2 was decreased. After the treatment of 1 nmol /L dihydrotestosterone and 10 nmol/L dihydrotestosterone for 2, 4 and 6 d, the cell proliferation inhibited degree of AR CK5 +CK8 + cells was higher compared with that of V CK5 +CK8 + cells, and the difference was statistically significant (all P < 0.05). After the treatment of 1 nmol/L dihydrotestosterone and 10 nmol /L dihydrotestosterone for 3 d, the migration ability of AR CK5 +CK8 + cells was decreased compared with that of V CK5 +CK8 + cells (the number of cell migration 54±9 vs. 113±21, 13±3 vs. 34±6), and the differences were statistically significant ( t=4.450, P<0.01; t=5.157, P<0.01).After the treatment of 1 nmol /L dihydrotestosterone and 10 nmol /L dihydrotestosterone for 3 weeks, the tumorigenic ability of AR CK5 +CK8 + cells was reduced compared with that of V CK5 +CK8 + cells (the number of clone 39±7 vs. 105±16, 41±6 vs. 86±6), and the differences were statistically significant ( t=6.631, P<0.01; t=8.662, P<0.01). And 5 nmol /L LY + 10 nmol/L 5-AZA, 5 nmol /L LY + 5 nmol/L γ-TT, 10 nmol/L 5-AZA + 5 nmol/L γ-TT, 2.5 nmol/L LY + 5 nmol/L 5-AZA + 2.5 nmol/L γ-TT combined with 1 nmol/L dihydrotestosterone or 10 nmol/L dihydrotestosterone after the treatment of 2, 4, 6 d inhibited the proliferation of CK5 +CK8 + cells (all P < 0.05). Conclusion:AR plays an inhibitory role in CK5 +CK8 + cells isolated from prostate cancer cell line LNCaP and reduces the cell migration and tumorigenic ability through inhibiting activation of AKT-bcl-2 signal pathway.