Impact of FTY720 on S1PR1-Positive HTLV-1-Infected T-Cell Lines
Yoshito Sadahira, Hideyo Fujiwara, Hirotake Nishimura, Takashi Akiyama, Isao Irei, Shuji Hamazaki. Kawasaki Medical School, Kurashiki, Japan
Background: FTY720, a sphingosine analog, is taken up by cells, phosphorylated to FTY720-phosphate (FTY720-P) by SK2, and then released. FTY720-P activates sphingosine-1-phosphate (S1P) receptors, mainly S1PR1, in a fashion similar to endogenous S1P. Despite the therapeutic potential of FTY720 for lymphohematological malignancies, the mechanism underlying the action of FTY720 is still unclear. In this study, we evaluated the ex vivo effects of FTY720 on HTLV-1-infected T-cell lines.
Design: Immunohistochemistry, immunocytochemistry, and Western blotting for S1PR1, STAT3, Akt, and PP2A, and quantitative real-time RT-PCR on S1PR were performed involving adult T-cell leukemia/lymphoma (ATLL) cases and various HTLV-1-infected T-cell lines including MT2. The effects of FTY720 and FTY720-P on cell proliferation and migration stimulated by serum were assessed using Cell Counting Kit-8 (Dojindo) and the Transwell-based migration assay, respectively.
Results: Immunohistochemistry for S1PR1 revealed positive membranous staining in 20% of primary ATLL. Real-time RT-PCR of MT2 cells showed higher levels of S1PR1 and S1PR4 than S1PR2 and extremely low levels S1PR3 and S1PR5. In contrast to FTY720-P, 10 μM of FTY720 inhibited the serum-induced cell proliferation of MT2 and other HTLV-1 infected S1PR1-negative T-cell lines. The high concentration of FTY720 inhibited the phosphorylation of Akt-pS473 and phosphorylation and nuclear immunostaining of STAT-pY705 of MT2 cells. On the other hand, the serum-induced migration of MT2 was inhibited by treatment with both FTY720 and FTY720-P even at 1 nM concentration for 30 min, and the inhibitory effect of FTY720 was delayed by 10 min compared to that of FTY720-P, suggesting that FTY720 inhibits migration via its conversion to FTY720-P. Treatment with 10 nM FTY720-P induced the phosphorylation of Akt-pS473 and PP2A-Y307, and S1PR1 internalization and degradation of MT2 within 10 min, while 10 nM FTY720 did not show those effects for 30 min. Pretreatment with W146 (S1PR1 antagonist) did not block the serum-induced migration of MT2 and FTY720-P's inhibition of this migration.
Conclusions: FTY720 may show an inhibitory effect on ATLL cell growth at high concentrations regardless of the expression of S1PRs. Even at very low concentrations, however, FTY720 may inhibit ATLL dissemination independently of S1PR1 internalization. Thus, our results have implications regarding the potential therapeutics of FTY720 in ATLL.
Wednesday, March 21, 2012 9:30 AM
Poster Session V # 193, Wednesday Morning