Investigating the Role of Guanosine on Human Neuroblastoma Cell Differentiation and the Underlying Molecular Mechanisms

Abstract

Neuroblastoma arises from neural crest cell precursors failing to complete the process of differentiation. Thus, agents helping tumor cells to differentiate into normal cells can represent a valid therapeutic strategy. Here, we evaluated whether guanosine (GUO), a natural purine nucleoside, which is able to induce differentiation of many cell types, may cause the differentiation of human neuroblastoma SH-SY5Y cells and the molecular mechanisms involved. We found that GUO, added to the cell culture medium, promoted neuron-like cell differentiation in a time- and concentration-dependent manner. This effect was mainly due to an extracellular GUO action since nucleoside transporter inhibitors reduced but not abolished it. Importantly, GUO-mediated neuron-like cell differentiation was independent of adenosine receptor activation as it was not altered by the blockade of these receptors. Noteworthy, the neuritogenic activity of GUO was not affected by blocking the phosphoinositide 3-kinase pathway, while it was reduced by inhibitors of protein kinase C or soluble guanylate cyclase. Furthermore, the inhibitor of the enzyme heme oxygenase-1 but not that of nitric oxide synthase reduced GUO-induced neurite outgrowth. Interestingly, we found that GUO was largely metabolized into guanine by the purine nucleoside phosphorylase (PNP) enzyme released from cells. Taken together, our results suggest that GUO, promoting neuroblastoma cell differentiation, may represent a potential therapeutic agent; however, due to its spontaneous extracellular metabolism, the role played by the GUO-PNP-guanine system needs to be further investigated.

Keywords: SH-SY5Ydifferentiation; guanine; guanosine; guanylate cyclase; heme oxygenase; neuroblastoma; protein kinase C; purine nucleoside phosphorylase.

FIGURE 1

Guanosine induces SH-SY5Y neuroblastoma cell differentiation in a dose- and time-dependent manner. (A) For the concentration-response curve, increasing concentrations (1–300 µM) of GUO were added for 48 h in medium containing 1% FBS. A non-linear regression analysis was performed and Emax and EC50 were calculated. (B) Representative images of SH-SY5Y cells cultures treated with 0, 50, and 100 μM GUO in medium with 1% FBS after 48 h. (C) For the time-dependent curve, cell cultures were incubated with GUO (50–100 µM) for different times (0–96 h) in a medium culture containing 1% FBS. In all cases, the number of differentiated cells, defined as cells having neurites more than one cell body length, was determined using a upright Zeiss Axiovert 200 microscope, total magnification 200x (Zeiss, Jena, Germany). Values are expressed as a percentage of neurite-bearing cells vs untreated cells and represent the mean ± SEM from at least three to five independent experiments.

FIGURE 2

Guanosine increases the expression of specific mature neuronal markers in SH-SY5Y neuroblastoma cells. SH-SY5Y cells were cultured for 48 h without treatment (a) or in the presence of 100 µM GUO (b) or 10 µM RA (c). βIII-Tubulin and MAP2 filaments are highlighted in green while nuclei are stained in blue with DAPI. For NeuN expression, on the left side of the image nuclei are stained in green with NeuN while on the right side of the image nuclei are stained in blue with DAPI. Images are representative of one of three independent experiments.

FIGURE 3

Mechanisms involved in guanosine-induced SH-SY5Y neuroblastoma cell differentiation. (A) Nucleoside transporter blockers (10 µM NBTI plus 100 µM propentofylline–PPF-plus 10 µM dypiridamole -DIP-) were simultaneously added to the medium containing 1% serum 1 h before GUO (100 µM) treatment and until the end of the experiment (48 h), while the antagonist of A₁ and A_2A adenosine receptor, DPCPX (100 nM) and ZM241385 (50 nM), respectively, were added 30 min before and until the end of the 48 h GUO (100 µM) treatment period. (B) The selective inhibitors of PI3K (25 µM LY294002), PKC (1 µM GF109203X), and sGC (10 µM OQD) or (C) the selective inhibitors of NOS (5 μM L-NAME), HO (1 µM ZnPP) and sGC (10 µM OQD) were added to cultures before the addition of GUO (100 µM) and during all the GUO treatment. In all cases, after 48 h, the total number of neurite-bearing cells was determined. Each value represents the mean ± SEM of at least five independent experiments in duplicate and is expressed as a percentage of neurite-bearing cells vs. untreated cells. Statistical analysis was performed using one-way ANOVA with the Dunnett’s or Turkey’s post-hoc multiple comparisons test; *p < 0.05, **p < 0.02, ****p < 0.001 compared with untreated cells; #p < 0.05, # #p < 0.02, # # #p < 0.01 compared with GUO treated cells.

FIGURE 4

Metabolic fate of extracellular guanosine and purine nucleoside phosphorylase (PNP) activity in SH-SY5Y cell cultures. (A) GUO (100 µM) was added to the culture medium containing 0% serum. At the indicated times, an aliquot of the medium was taken and analyzed by HPLC as described in materials and methods. (B) PNP activity was measured within SH-SY5Y neuroblastoma cells or (C) in the culture medium. For this purpose, SH-SY5Y cells were incubated in a serum-free medium supplemented or not with 100 µM GUO. After 6 and 24 h, an aliquot of the medium was taken and the enzyme present was concentrated using Amicon Ultra filters while cells were scraped in lysis buffer and cytosolic extracts were prepared. PNP activity was assayed using 100 μM GUO as substrate plus 50 mM Pi as co-substrate for 15 min at 37°C. The concentration of the newly formed product, GUA, was measured by HPLC analysis. PNP activity was expressed as milli-International Units (mIU) per mg protein or ml of culture medium. Values are the mean ± SEM of at least five independent experiments, each run in duplicate. Statistical analysis was performed using a two-way ANOVA followed by Turkey’s multiple comparison test, *p < 0.05 compared with untreated cells at 6 h and #p < 0.05 compared with GUO treated cells at 6 h.