Journal of Cellular Biochemistry. 2017 June 6. doi:10.1002/jcb25798

Increase of Intracellular Cyclic AMP by PDE4 Inhibitors Affects HepG2 Cell Cycle Progression and Survival

Massimi M1, Cardarelli S2, Galli F1, Giardi MF1, Ragusa F1, Panera N3, Cinque B1, Cifone MG1, Biagioni S2, Giorgi M2.

1Department of Life, Health and Environmental Sciences, University of L’Aquila, L’Aquila, Italy.

2Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Rome, Italy.

3Liver Research Unit, Bambino Gesù Children’s Hospital and IRCC, Rome, Italy.

Correspondence should be addressed to Massimi Mara, Department of Life, Health and Environmental Sciences, University of L’Aquila, Italy, E.mail: mara.massimi@univaq.it

 

Abstract

Type 4 cyclic nucleotide phosphodiesterases (PDE4) are major members of a superfamily of enzymes (PDE) involved in modulation of intracellular signaling mediated by cAMP. Broadly expressed in most human tissues and present in large amounts in the liver, PDEs have in the last decade been key therapeutic targets for several inflammatory diseases. Recently, a significant body of work has underscored their involvement in different kinds of cancer, but with no attention paid to liver cancer. The present study investigated the effects of two PDE4 inhibitors, rolipram and DC-TA-46, on the growth of human hepatoma HepG2 cells. Treatment with these inhibitors caused a marked increase of intracellular cAMP level and a dose- and time-dependent effect on cell growth. The concentrations of inhibitors that halved cell proliferation to about 50% were used for cell cycle experiments. Rolipram (10 μM) and DC-TA-46 (0.5 μM) produced a decrease of cyclin expression, in particular of cyclin A, as well as an increase in p21, p27 and p53, as evaluated by Western blot analysis. Changes in the intracellular localization of cyclin D1 were also observed after treatments. In addition, both inhibitors caused apoptosis, as demonstrated by an Annexin-V cytofluorimetric assay and analysis of caspase-3/7 activity. Results demonstrated that treatment with PDE4 inhibitors affected HepG2 cell cycle and survival, suggesting that they might be useful as potential adjuvant, chemotherapeutic or chemopreventive agents in hepatocellular carcinoma.

KEYWORDS: apoptosis; cyclic nucleotide phosphodiesterase; cyclins; DC-TA-46; human hepatocarcinoma cells; HEPARG; liver; rolipram

 

Supplement:

Hepatocellular carcinoma (HCC) is the most common primary malignant disease of the liver. The main risk factors for HCC are obesity, alcohol abuse and viral infection (HBV and HCV). Environmental exposure to toxins such as aflatoxin B1 and diethylnitrosamine also contributes to its development [1]. Since a reliable biomarker is still lacking, diagnosis of HCC often arrives when the cancer is in a late stage. At present, the election therapy for this cancer is sorafenib, a multikinase inhibitor with pleiotropic effects [2], but chemotherapy response rates are low and at present mortality remains very high.

cAMP is a second messenger that can open ion channels regulated by cyclic nucleotides and activate PKA (protein kinase activated by cAMP). The targets of PKA include receptors, cytoskeletal proteins and transcription factors, therefore cAMP may regulate many important cellular functions, including proliferation and differentiation. Levels of cAMP are regulated by two classes of enzymes: adenylate cyclase, which promotes its synthesis, and phosphodiesterases (PDE), which promote its degradation. PDEs are a class of enzymes divided into 11 families based on sequence homology, sensitivity to specific inhibitors and, especially, substrate specificity (cAMP or cGMP). Some families hydrolyze only cAMP or cGMP, while others are able to hydrolyze both. Many studies in the literature demonstrate that different types of cancer show decreased intracellular cAMP levels and higher levels of phosphodiesterase protein and activity [3] compared to normal tissues, but little data is available for HCC.

In this paper, we investigated the role of cAMP signaling in the human hepatocellular carcinoma cells, HepG2, focusing on phosphodiesterase-4 (PDE4) and using two selective inhibitors: rolipram, a widely used PDE4 inhibitor, and DC-TA-46, which has been much less studied so far, with only few data present in the literature. Both inhibitors are characterized by elevated activity and low toxicity, and are already in use for several clinical applications with good tolerability.

We verified first the presence of PDE activity in the proliferating HepG2 cells as well as in the non-tumorigenic, non-proliferating HepaRG cells and primary rat hepatocytes. Data has shown higher levels of cAMP phosphodiesterase activity in HepG2 cells compared with both HepaRG and primary rat hepatocytes[4]. We then used MonoQ ion exchange chromatography to investigate the pattern of phosphodiesterase isoforms in HepG2 cells. The elution profile displayed five peaks of activity – peak IV represented about 45% of the total cAMP activity. Peak IV was also three times higher than in the non-proliferating, differentiated primary rat hepatocytes described in the literature under similar conditions and with a similar elution profile. In addition, this fraction was highly sensitive to both rolipram and DC-TA-46. Western blot analysis using antibodies specific for all the PDE4 isoforms (PDE4A, PDE4B, PDE4C and PDE4D) confirmed a higher activity of PDE4 in HepG2 compared to the non-tumorigenic cells [4].

We next analyzed cell viability and inhibitor cytotoxicity and found that while rolipram had a moderate effect, DC-TA-46 had a marked effect on HepG2 cells [4]. We then conducted flow cytometric analysis after double staining with AnnexinV/propidium iodide to determine whether the decreased number of cells was mainly due to apoptosis or necrosis. Results showed that both inhibitors were able to induce apoptosis, with more significant results obtained with DC-TA-46 [Figure 1A]. In addition, analysis of cell cycle distribution showed that inhibitors could significantly slow down the HepG2 cell cycle, but while rolipram induced cell accumulation in S phase, DC-TA-46 blocked the cells at the earliest stage, G1, thus explaining the increased cell death by apoptosis after DC-TA-46 treatment [Figure 1B].

We also analyzed the protein expression of key cell cycle molecules – PDE4 inhibitors induced a significant deregulation of cyclin A and cyclin B1, while cyclin D1 decreased appreciably only with DC-TA-46 [Figure 1C]. Nonetheless, a delocalization from nucleus to cytoplasm of this cyclin was observed after treatment with rolipram [4]. In addition, the levels of inhibitors of cyclin-cdk complexes, p21 and p27, increased with both inhibitors, although with a dissimilar trend [Figure 1D]. The greater efficiency of DC-TA-46, although difficult to interpret, could be related to a wider spectrum of action of this drug that could affect other signalling pathways, including those mediated by cGMP. A certain degree of DC-TA-46 activity was actually demonstrated towards PDE5, PDE1 and PDE2 [Drees et al. 1993]. A differential effect on the different PDE4 isoforms cannot be excluded, but at this time there are still no studies available on this topic, although it is of great interest.

In conclusion, our work clearly shows that PDE4 inhibitors are able to reverse the tumor phenotype of hepatocytes, at least partially, in agreement with data present in the literature that show their efficacy in other types of cancer, and thus they should be considered as adjuvant drugs for the treatment of HCV, particularly those that are refractory to existing therapies. Synergistic effects with chemotherapy drugs currently in use deserve further studies and are currently underway in our laboratory.

 

 

Figure 1 Effect of 0.5 μM DC-TA-46 and 10 μM rolipram on HepG2 cells. (A) Apoptosis analysis. Cells were stained with annexin V/propidium iodide and treated for 48h or 72h. (B) Cell cycle analysis. Cell population distribution in the cell cycle phases analyzed by flow-cytometry after 48h of treatment. (C) Western blot quantitative analysis of cyclin D1, cyclin A and cyclin B1 after 48h of treatment. (D) Western blot quantitative analysis of p53, p21 and p27 after 48h of treatment. All results are the mean ± standard deviation of at least three different experiments. *P<0.05; **P<0.005.

 

 

References

1 Bodzin AS, Busuttil RW. 2015. Hepatocellular carcinoma: Advances in diagnosis, management, and long term outcome. World J Hepatol. 7 :1157-67.

2Moriguchi M, Umemura A, Itoh Y. 2016. Current status and future prospects of chemotherapy for advanced hepatocellular carcinoma. Clin J Gastroenterol. 9:184-90.

3Savai R, Pullansetti SS, Banat GA, Weismann N, Ghofram H, Grimminger F, Schermily R. 2010. Targeting cancer with phosphodiesterase inhibitors. Expert opinion on investigational drugs; 19: 117-131.

4Massimi M, Cardarelli S, Galli F, Giardi MF, Ragusa F, Panera N, Cinque B, Cifone MG, Biagioni S, Giorgi M. 2017. Increase of Intracellular Cyclic AMP by PDE4 Inhibitors Affects HepG2 Cell Cycle Progression and Survival. Journal of Cellular Biochemistry; 118:1401-1411.