Inhibition of isoform D of phosphodiesterase type 4 reduces cell proliferation and survival in hepatocarcinoma cell lines

Cyclic AMP phosphodiesterases (PDEs) are enzymes that regulate intracellular levels of cAMP by controlling its rate of degradation, in contrast to adenylyl cyclases that synthesize cAMP from intracellular ATP. PDEs are classified in 11 families (PDE1-PDE11). Some of these control the intracellular levels of both cyclic nucleotides, while others (PDE4, 7, 8) are specific for cAMP degradation. Previous experiments showed that isoform D of cAMP specific PDE4 (PDE4D) was aberrantly up-regulated in HCC cells, in particular in the highly tumorigenic Hep3B and Huh7 cell lines, which also showed increased PDE4 activity. In agreement, tumor tissues displayed stronger staining for the PDE4D isoform when compared to normal liver tissues. The aim of the present study was to analyze the effects of inhibition of this isoform with the final goal of understanding its role in the development of hepatocellular carcinoma and shedding light on the mechanisms eventually involved in this process. SiRNA-mediated transient silencing of PDE4D expression reduced cell proliferation and triggered apoptosis, with an increased number of cells found in the G0/G1 phase of the cell cycle, as shown by DELFIA proliferation assay and flow cytometry experiments. In agreement, Western blot experiments revealed inhibition of cyclin D1, as well an increase in p21, p27 and p53 protein expression. The pharmacological selective inhibition of PDE4D with Gebr-7b gave similar results, with no toxicity. In conclusion, this study established a crucial role of PDE4D in the hepatic tumor phenotype. In addition to being a biomarker for diagnosis, PDE4D could represent a specific adjuvant therapeutic target for the treatment of hepatocellular carcinoma, in particular for those cases that are refractory to existing therapies