In Vitro Effects of a Phosphodiesterase Inhibitor Sildenafil on Cellular Motility of the Oral Malignant Melanoma Cells
Human oral malignant melanoma MAA cells were established and maintained in our laboratory.
Cell viability assay. MAA cells (5×104cells/well) were plated in 24 wells and cultured for 24 hours. Then, the cells were treated with sildenafil for 3 days (5 μM and 10 μM). Trypan Blue Stain at 0.4% was added to the cells and both living and dead cells were counted under a microscope.
In vitro invasion assays. MAA cells (1×104cells) in RPMI 1640 medium containing 0.1% FBS were transferred to 8 mm pore Matrigel pre-coated inserts with or without sildenafil. The inserts were placed in companion wells containing medium supplemented with 10% FBS as a chemoattractant. Following 5 hours incubation, the non-invading cells were removed from the upper surface of the membrane. The cells on the lower surface of the membrane were fixed, stained with Diff‑Quik, and counted under a microscope.
In vitro migration assay.The procedures were basically same as the above invasion assay except that simple 8-μm pore inserts were used instead of the Matrigel-coated inserts. The effects of Ht-31 on cell migration were assessed by the number of the cells on the lower surface of the membrane.
The differences among multiple groups were analyzed using Tukey-Kramer multiple comparison test, and the difference between two groups were analyzed using Student's t-test.
As the results, sildenafil at the concentrations used in this study did not show any significant effects on cell viability and invasion of the MAA cells. On the other hand, Ht31 inhibited migration of the MAA cells.
The present results showed that sildenafil could not inhibit the invasion of MAA cells, although our previous study had demonstrated that sildenafil suppresses migration of these cells. Ht-31, a PKA inhibitor, inhibited cell migration in this study. As it is known that PKA exists downstream the PDE-cAMP signaling, it is suggested that sildenafil can affect PDE signaling and cell motility, but not cell invasion which needs degradation of matrix. Due et al.2), have reported that PKA is a key regulator of Rho families. So, further studies on PDE, PKA and Rho family are necessary to expect the possibility of sildenafil for inhibiting metastasis of melanoma cells.
1. Joseph, B.A., Sharron, F.H., et al.: Cyclic Nucleotide Phosphodiesterases in Health and Disease. First Edition pp.1-17, CRC Press Taylor & Francis Group, the United States of America, 2006.
2. Dua P, Gude RP. Pentoxifylline impedes migration in B16F10 melanoma by modulating Rho GTPase activity and actin organisation. Eur J Cancer 44(11):1587-95, 2008