Role of phosphodiesterase 2 in growth and invasion of human malignant melanoma cells

Abstract

Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentrations and effects of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP). The role of PDEs in malignant tumor cells is still uncertain. The role of PDEs, especially PDE2, in human malignant melanoma PMP cell line was examined in this study. In PMP cells, 8-bromo-cAMP, a cAMP analog, inhibited cell growth and invasion. However, 8-bromo-cGMP, a cGMP analog, had little or no effect. PDE2 and PDE4, but not PDE3, were expressed in PMP cells. Growth and invasion of PMP cells were inhibited by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a specific PDE2 inhibitor, but not by rolipram, a specific PDE4 inhibitor. Moreover, cell growth and invasion were inhibited by transfection of small interfering RNAs (siRNAs) specific for PDE2A and a catalytically-dead mutant of PDE2A. After treating cells with EHNA or rolipram, intracellular cAMP concentrations were increased. Growth and invasion were stimulated by PKA14-22, a PKA inhibitor, and inhibited by N(6)-benzoyl-c AMP, a PKA specific cAMP analog, whereas 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, an Epac specific cAMP analog, did not. Invasion, but not growth, was stimulated by A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide. Based on these results, PDE2 appears to play an important role in growth and invasion of the human malignant melanoma PMP cell line. Selectively suppressing PDE2 might possibly inhibit growth and invasion of other malignant tumor cell lines.

Keywords: Cell growth; Cyclic AMP; Human malignant melanoma; Invasion; Phosphodiesterase 2.

Fig. 1

Effects of 8-Br-cAMP or 8-Br-cGMP on cell growth and invasion. Cell growth was measured using the MTS assay. Cells were cultured in the absence or presence of 8-Br-cAMP (0.1 to 1 mM) or 8-Br-cGMP (0.1 to 1 mM) for 5 days. Cell invasion was examined by in vitro Matrigel invasion assays. Cells were transferred to 8 µm pore Matrigel pre-coated inserts, and 8-Br-cAMP (0.1 to 1 mM) or 8-Br-cGMP (0.1 t 1 mM) was added. After a 16 h incubation, invaded cells were stained with May-Grünwald-Giemsa stain and counted. Data in graphs are means of three independent experiments, each performed in duplicate. (A) Effect of 8-Br-cAMP on cell growth. (B) Effect of 8-Br-cAMP on cell invasion. (C) Effect of 8-Br-cGMP on cell growth. (D) Effect of 8-Br-cGMP on cell invasion. The error bars represent means ± SD, n = 3. The treatments that differ significantly from control are noted (*, P < 0.01).

Fig. 2

Expression of PDEs and effects of 8-Br-cAMP on PDE activity in PMP cells. Data in graphs are means of three independent experiments, each performed in triplicate. (A) PDE activities were analyzed by cAMP PDE activity assay with or without each specific PDE inhibitor. The error bars represent means ± SD (n = 3). The concentrations of each reagents were: EHNA, 20 µM; cGMP, 10 µM; cilostamide, 0.5 µM; rolipram, 10 µM. (B) Effect of 8-Br-cAMP on cGMP-stimulated PDE activity in PMP cells. cGMP (10 µM) and 8-Br-cAMP (0.1 to 1 mM) were used. The error bars represent means ± SD, n = 3. (C) Effect of 8-Br-cAMP with or without rolipram on PDE activity in PMP cells. Rolipram (10 µM) and 8-Br-cAMP (0.1 to 1 mM) were used. (D) Expression of PDE mRNAs in PMP cells. RT-PCR analysis for PDE2, PDE3, and PDE4 mRNAs were performed. HMG cells derived from human gingival malignant melanoma were used as the positive control (PC) for PDE3A, 3B, and 4D mRNAs. Experiments were repeated three times, and similar results were obtained. 2A = PDE2A; 3A = PDE3A; 3B = PDE3B; 4A = PDE4A; 4B = PDE4B; 4C = PDE4C; 4D = PDE4D; M = molecular markers.

Fig. 3

Western blotting of PDE3s and PDE4s. Experiments were repeated two times, and similar results were obtained. (A) Western blotting of PDE3A. Positive control (P.C.) was human full-length PDE3A recombinant protein. (B) Western blotting of PDE3B. Positive control (P.C.) was human PDE3B recombinant protein from Signal Chem.. (C) Western blotting of PDE4A. Positive control (P.C.) was KB cells. (D) Western blotting of PDE4B. (E) Western blotting of PDE4C. (F) Western blotting of PDE4D. Positive control (P.C.) was human PDE4D recombinant protein from Signal Chem.. The positions of marker proteins and their sizes in kDa are given left on each panel.

Fig. 4

Effect of PDE2A siRNA transfection (5 nM of siRNA). Data in graphs are means of three independent experiments, each performed in triplicate. (A) Expression levels of PDE2A mRNA analyzed by quantitative real-time PCR. The error bars represent means ± SD, n = 3. A significant treatment effect is noted (*, P < 0.01 compared with control siRNA treatment). (B) Expression of PDE2 protein analyzed by western blotting. Experiments were repeated three times, and similar results were obtained. (C) Effect of PDE2A siRNA on PDE activity in PMP cells. The concentrations of each reagents were: EHNA, 20 µM; cGMP, 10 µM; cilostamide, 0.5 µM; rolipram, 10 µM. (D) Effect of 8-Br-cAMP on PDE2 A siRNA inhibited PDE activity in PMP cells. cGMP (10 mM) and 8-Br-cAMP (0.1 to 1 µM) were used.

Fig. 5

Effects of EHNA, rolipram, or PDE2A siRNA transfection on cell growth. Data in graphs are means of three independent experiments, each performed in triplicate. (A) Effect of EHNA on cell growth. PMP cells were cultured in the absence or presence of EHNA (1 to 100 µM) for 5 days. The error bars represent means ± SD, n = 3. The treatments that differ significantly from control are noted (*, P < 0.01 ). (B) Effect of PDE2A siRNA on cell growth. Cells were transfected with 5 nM siRNA, and, after 48 h, plated at 400 cells/well in a 96-well plate. The error bars represent means ± SD, n = 3. A significant treatment effect is noted (*, P < 0.01 compared with control siRNA treatment). (C) Effect of rolipram on cell growth. Five days after the addition of rolipram, the number of cells was measured. The error bars represent means ± SD, n = 3. (D) Combined effects of EHNA and rolipram on cell growth. Five days after addition of EHNA (1, 10 µM) and rolipram (10, 50 µM), the number of cells was measured.

Fig. 6

Effects of EHNA, rolipram, or PDE2A siRNA transfection on cell invasion. Data in graphs are means of three independent experiments, each performed in triplicate. (A) Effect of EHNA on cell invasion examined by in vitro Matrigel invasion assays. The error bars represent means ± SD, n = 3. The treatments that differ significantly from control are noted (*, P < 0.01). (B) Effect of PDE2A siRNA on cell invasion. Cells were transfected with 5 nM siRNA, and cell invasion was examined after 2 days. The error bars represent means ± SD, n = 3. A significant treatment effect is noted (*, P < 0.01 compared with control siRNA treatment). (C) Effect of rolipram on cell invasion. The error bars represent means ± SD, n = 3.

Fig. 7

Effect of EHNA and rolipram on intracellular cAMP content. PMP cells were incubated with medium containing EHNA (10 and 50 µM) or rolipram (10 and 50 µM) for 15 min, and intracellular cAMP content was determined using the enzyme immunoassay kit. Data are means of three independent experiments, each performed in triplicate. The error bars represent means ± SD, n = 3. A significant treatment effect is noted (*, P < 0.01 compared with control).

Fig. 8

Effects of mRFP-tagged dnPDE2A (catalytically dead mutant) on cell growth and invasion. Data in graphs are means of three independent experiments, each performed in triplicate. (A) Effects of mRFP-tagged dnPDE2A on cell growth. Cells were transfected with mRFP-tagged dnPDE2A, and, after 48 h, plated at 400 cells/well in a 96-well plate. The error bars represent means ± SD, n = 3. A significant treatment effect is noted (*, P < 0.01 compared with Mock treatment). (B) Effects of mRFP-tagged dnPDE2A on cell invasion. Cells were transfected with mRFP-tagged dnPDE2A, and cell invasion was examined after 16 h. The error bars represent means ± SD, n = 3. A significant treatment effect is noted (*, P < 0.01 compared with mock treatment).

Fig. 9

Immunocytochemistry of PDE2A. Immunocytochemistry was performed as described in Material and methods. Experiments were repeated two times, and similar results were obtained.

Fig. 10

Effects of PKA related reagents and 8-pCPT-2’-O-Me-cAMP on cell growth. Cells were cultured in each reagent for 5 days. Cell growth was analyzed by MTS assay. (A) Effect of PKI_14–22 on cell growth. (B) Effect of N⁶-Benzoyl-cAMP on cell growth. (C) Effect of HT-31 on cell growth. (D) Effect of 8-pCPT-2’-O-Me-cAMP on cell growth. Data in graphs are means of three independent experiments, each performed in triplicate. The error bars represent means ± SD, n = 3. The treatments that differ significantly from control are noted (*, P < 0.01).

Fig. 11

Effects of PKA related reagents and 8-pCPT-2’-O-Me-cAMP on cell invasion. Cell invasion was examined by in vitro Matrigel invasion assays. Cells were transferred to 8 µm pore Matrigel pre-coated inserts, and each reagents was added. After a 16 h incubation, invaded cells were stained with Diff-Quik™ and counted. Data in graphs are means of three independent experiments, each performed in triplicate. (A) Effect of PKI_14–22 on cell invasion. (B) Effect of N⁶-Benzoyl-cAMP on cell invasion. (C) Effect of HT-31 on cell invasion. (D) Effect of 8-pCPT-2’-O-Me-cAMP on cell invasion. The error bars represent means ± SD, n = 3. The treatments that differ significantly from control are noted (*, P < 0.01).