Intracellular cGMP increase is not involved in thyroid cancer cell death

Abstract

Introduction: Type 5 phosphodiesterase (PDE5) inhibitors (PDE5i) lead to intracellular cyclic-guanosine monophosphate (cGMP) increase and are used for clinical treatment of erectile dysfunction. Studies found that cGMP may up/downregulate the growth of certain endocrine tumor cells, suggesting that PDE5i could impact cancer risk.

Aim: We evaluated if PDE5i may modulate thyroid cancer cell growth in vitro.

Materials and methods: We used malignant (K1) and benign (Nthy-ori 3-1) thyroid cell lines, as well as the COS7 cells as a reference model. Cells were treated 0-24 h with the PDE5i vardenafil or the cGMP analog 8-br-cGMP (nM-μM range). cGMP levels and caspase 3 cleavage were evaluated by BRET, in cGMP or caspase 3 biosensor-expressing cells. Phosphorylation of the proliferation-associated extracellularly-regulated kinases 1 and 2 (ERK1/2) was evaluated by Western blotting, while nuclear fragmentation by DAPI staining. Cell viability was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Results: Both vardenafil and 8-br-cGMP effectively induced dose-dependent cGMP BRET signals (p≤0.05) in all the cell lines. However, no differences in caspase 3 activation occurred comparing PDE5i-treated vs untreated cells, at all concentrations and time-points tested (p>0.05). These results match those obtained upon cell treatment with 8-br-cGMP, which failed in inducing caspase 3 cleavage in all the cell lines (p>0.05). Moreover, they reflect the lack of nuclear fragmentation. Interestingly, the modulation of intracellular cGMP levels with vardenafil or the analog did not impact cell viability of both malignant and benign thyroid tumor cell lines, nor the phosphorylation of ERK1/2 (p>0.05).

Conclusions: This study demonstrates that increased cGMP levels are not linked to cell viability or death in K1 and Nthy-ori 3-1 cell lines, suggesting that PDE5i do not impact the growth of thyroid cancer cells. Since different results were previously published, further investigations are recommended to clarify the impact of PDE5i on thyroid cancer cells.

Fig 1. Analysis of intracellular cGMP levels.

Transfected cells expressing the cGMP/Rluc-tagged biosensor were treated 2 h with increasing 8-Br-cGMP and vardenafil (fM-mM range). BRET signals indicating intracellular cGMP levels were evaluated and plotted against the concentration of inhibitor/analog. A, B) Data from the COS7 cell line treated with 8-Br-cGMP and vardenafil, respectively; C, D) 8-Br-cGMP- and vardenafil-treated K1 cell line; E, F) Nthy-ori 3–1 cells treated with 8-Br-cGMP and vardenafil. Results were compared by Kruskal Wallis test and Dunn’s post-test versus untreated samples and statistically significant differences were marked by * (p<0.05; means ± SEM; n = 8 to 15).

Fig 2. Modulation of cell viability by 8-Br-cGMP/vardenafil.

COS7, K1 and Nthy-ori 3–1 cell lines were treated 24 h with increasing 8-Br-cGMP and vardenafil (100 nM– 500 μM range). Cell viability was evaluated by MTT assay and indicated as an absorbance detected at the 570 nm wavelength. 4 μM thapsigargin served as a positive control (Tha) for cell viability decrease and results were compared by Kruskal Wallis test and Dunn’s post-test (*significantly different versus untreated sample; p < 0.05; means ± SEM; n = 6 to 8). A, B) Data from the COS7 cell line treated with 8-Br-cGMP and vardenafil, respectively; C, D) Results from treatments of K1 cells; E, F) Nthy-ori 3–1 cell line.

Fig 3. 15-min, 8-Br-cGMP/vardenafil-induced ERK1/2 phosphorylation in the three cell lines.

Cells were treated as follow: a) untreated; b) 10 μM 8-Br-cGMP; c) 50 μM vardenafil; d) preincubation with NS 2028 (GCi) and treatment with 8-Br-cGMP; e) preincubation with NS 2028 (GCi) and treatment with vardenafil; f) NS 2028 (GCi); g) MEK inhibitor U0126 was used to deplete ERK1/2 phosphorylation h) PMA was used as positive control of pERK1/2. Data from densitometric analysis were compared by Kruskal Wallis test and Dunn’s post-test (*significantly different versus untreated samples; p ≥ 0.05; means ± SEM were superimposed to data points; n = 3).

Fig 4. Caspase 3 activity in 8-Br-cGMP/vardenafil-treated COS7, K1 and Nthy-ori 3–1 cell lines.

The activity of the enzyme was detected by BRET, in cells treated 4 h with increasing 8-Br-cGMP and vardenafil concentrations (100 nM– 500 μM range) and expressing a specific biosensor. 4 μM thapsigargin served as a positive control (Tha). Results were compared by Kruskal Wallis test and Dunn’s post-test (*significantly different versus untreated sample; p < 0.05; means ± SEM; n = 15). A, B) 8-Br-cGMP- and vardenafil-treated COS7 cells, respectively; C, D) Results from K1 cells; E, F) Nthy-ori 3–1 cell line.

Fig 5. Evaluation of nuclear damage.

COS7, K1 and Nthy-ori 3–1 cells were treated 24 h with 1 μM 8-Br-cGMP and 50 μM vardenafil before to be fixed and DAPI stained. 4 μM thapsigargin-treated cells served as positive controls. 60x magnification; scale bar = 10 μM; n = 1.