Effects of extracellular nucleotides and nucleosides on prostate carcinoma cells

Abstract

1. The purpose of this work was to characterize the receptors involved in the action of nucleotides on the human prostate carcinoma cell lines LNCaP, PC-3 and DU145. 2. Northern blotting revealed the presence of P2Y(2), P2Y(6) and P2Y(11) messengers in the three cell lines. P2Y(1) mRNA was only observed in the DU145 cells. In both PC-3 and DU145 cells, ATP and UTP stimulated inositol phosphate accumulation in an equipotent, equiactive and non-additive way, suggesting the involvement of P2Y(2) receptors. ATP also increased cyclic AMP, but this effect is likely to result from degradation into adenosine and activation of A(2) receptor. A(2) receptor activation led to a synergistic enhancement of prostate-specific antigen secretion induced by vasoactive intestinal peptide. 3. RT - PCR experiments detected the expression of the P2X(4) and P2X(5) receptors in the DU145 cells and the P2X(4), P2X(5) and P2X(7) receptors in the PC-3 cells. The calcium influx induced by BzATP confirmed the functional expression of P2X receptors. 4. ATP inhibited the growth of PC-3 and DU145 cells. This effect was mimicked neither by UTP nor by adenosine, indicating that it does not result from phospholipase C or adenylyl cyclase activation. On the contrary, in PC-3 cells, BzATP reproduced the effect of ATP, which was associated to a moderate decrease of proliferation and an increase of apoptosis. In DU145 cells, ATP was more potent than BzATP and growth inhibition was mainly associated with necrosis. We suggest that P2X receptors might be involved in the inhibition by nucleotides of prostate carcinoma cell growth.

Figure 1

Concentration-action curves of UTP and ATP on the InsP₃ accumulation in PC-3 or DU145 cells. ³H-inositol-labelled cells, preincubated 20 min with 10 mM LiCl, were incubated in the presence of various concentrations of nucleotides for 5 min (PC-3 cells) or 15 min (DU145 cells). The data are mean±s.e.mean of three independent experiments with triplicate experimental points.

Figure 2

Effects of various nucleotides and adenosine on the cAMP production in prostatic carcinoma cells. (a) The PC-3 cells, preincubated 30 min with rolipram 25 μM, were stimulated for 15 min with adenosine (1 μM), ATP (100 μM), ATPγS (100 μM), BzATP (100 μM), ADP (100 μM), adenosine+8-PST (300 μM), ATP+8-PST or without agonist (CONTROL) in a medium containing rolipram 25 μM. The data represent the mean±s.e.mean of three independent experiments with triplicate experimental points. (b) The DU145 cells, preincubated 30 min with rolipram 25 μM, were stimulated for 15 min with adenosine (1 μM), ATP (100 μM), ATPγS (100 μM), FK (1 μM), FK+adenosine, FK+ATP, FK+ATPγS, FK+adenosine+8-PST (100 μM) or FK+ATP+8-PST in a medium containing rolipram 25 μM. The data represent the mean±s.e.mean of three independent experiments with triplicate experimental points. (c) The LNCaP cells, preincubated 30 min with rolipram 25 μM, were stimulated for 15 min with adenosine (1 μM), ATP (100 μM), ATPγS (100 μM), FK (3 μM), FK+adenosine, FK+ATP, FK+ATPγS or FK+adenosine+8-PST in a medium containing rolipram 25 μM. The data represent the mean±s.e.mean of three independent experiments with triplicate experimental points.

Figure 3

Concentration-action curves of VIP or VIP+adenosine (10 μM) on the cyclic AMP accumulation and PSA secretion in LNCaP cells. 300,000 LNCaP cells were incubated for 2 h in a serum free RPMI 1640 medium containing the agonist or not (control). Extracellular medium was removed and PSA assayed using a MEIA procedure on an AXSYM device. Measurements of cyclic AMP were performed as described under Methods. Data are the mean±s.e.mean of three different experiments performed with triplicate experimental points.

Figure 4

Effects of ATP or UTP on growth of prostatic carcinoma cells. PC-3 cells, at a density of 50,000 cells/3.5 cm dishes, were seeded at day 0 in a complete Ham's F-12 medium containing 8% FBS, with or without the agonist (100 μM). Nucleotides were renewed each day, and medium changed at day 2. The number of adherent living cells that excluded trypan blue was determined at days 1, 3 and 4 using a haemocytometer. DU145 cells were seeded at a density of 50,000 cells/3.5 cm dishes in a complete Eagle's modified essential medium containing 4% FBS. LnCAP cells were seeded at a density of 100,000 cells/3.5 cm dishes in complete RPMI 1640 medium containing 10% FBS. Treatment and counting of DU145 and LNCaP are similar to those described for PC-3 cells. The data represent the mean±s.e.mean of three independent experiments with quadruplicate experimental points. ^*Indicates that the level of significance (P) between control and ATP conditions is P<0.005.

Figure 5

Effects of nucleotides, adenosine and/or FK on growth of prostatic carcinoma cells. Culture conditions are similar to those described in Figure 4. Cells were incubated in the presence or absence of nucleotides for 4 days and the number of living cells that excluded trypan blue was determined using a haemocytometer. Stimulations were performed with ATP (100 μM), UTP (100 μM), adenosine (100 μM), FK (10 μM) or 8-PST (300 μM for PC-3 cells and 100 μM for other cell lines alone or in combination). The data represent the mean±s.e.mean of three independent experiments with quadruplicate experimental points. ^*Indicates that the level of significance (P) between control and stimulated conditions is P<0.005.

Figure 6

Concentration-action curves of effects of various adenine nucleotides on androgen-independent prostatic cell growth. PC-3 and DU145 cells were cultured and counted in the same conditions as described in Figure 4. Cells were submitted or not to stimulation with BzATP, ATP, ATPγS, 2-MeSATP or α,βmeATP (all at 100 μM). After 4 days, the number of living cells that excluded trypan blue was determined using a haemocytometer. The data represent the mean±s.e.mean of three independent experiments with quadruplicate experimental points.

Figure 7

Qualitative detection of apoptosis among androgen-independent PC-3 and DU145 cells. PC-3 cells, at a density of 400,000/10 cm dishes, were cultured in complete Ham's F-12 containing 8% FBS and stimulated or not by BzATP or ATP (100 μM). DU145 cells, 400,000/10 cm dishes, were cultured in a complete Eagle's modified essential medium containing 4% FBS. Experimental procedures are described under Methods. DNA (8 μg/lane) was separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining.

Figure 8

Detection of P2X receptors mRNA in human androgen-independent prostatic carcinoma cells by RT–PCR experiments. The experimental procedure as well as oligonucleotide sequences are described under Methods. PCR products (P2X₄: 424 pb; P2X₅: 373 pb; P2X₇: 470 pb) are visualized after electrophoresis, and ethidium bromide staining of a 2% agarose gel. These data are representative of three different RT – PCR experiments, performed on two different batches of RNA.

Figure 9

Measurements of intracellular calcium using the fluorescent marker fura 2-AM. Effects of BzATP (A) and ATP (B) at 100 μM on the PC-3 cells, in a medium containing or not EGTA (1 mM) added just prior to the ligand stimulation. The experimental procedure is described under Methods. The data represent the mean of three independent experiments (s.e.means were below 10%).