The inhibitory effects of extracellular ATP on the growth of nasopharyngeal carcinoma cells via P2Y2 receptor and osteopontin
- Guang Yang†1,
- Shenghong Zhang†2,
- Yanling Zhang†3,
- Qiming Zhou1,
- Sheng Peng1,
- Tao Zhang1,
- Changfu Yang4,
- Zhenyu Zhu4 and
- Fujun Zhang1Email author
© Yang et al.; licensee BioMed Central Ltd. 2014
Received: 17 March 2014
Accepted: 17 June 2014
Published: 24 June 2014
Nasopharyngeal carcinoma (NPC) is a common malignant tumor observed in the populations of southern China and Southeast Asia. However, little is known about the effects of purinergic signal on the behavior of NPC cells. This study analyzed the effects of ATP on the growth and migration of NPC cells, and further investigated the potential mechanisms during the effects.
Cell viability was estimated by MTT assay. Transwell assay was utilized to assess the motility of NPC cells. Cell cycle and apoptosis were detected by flow cytometry analysis. Changes in OPN, P2Y2 and p65 expression were assessed by western blotting analysis or immunofluorescence. The effects of ATP and P2Y2 on promoter activity of OPN were analyzed by luciferase activity assay. The binding of p65 to the promoter region of OPN was examined by ChIP assay.
An MTT assay indicated that ATP inhibited the proliferation of NPC cells in time- and dose-dependent manners, and a Transwell assay showed that extracellular ATP inhibited the motility of NPC cells. We further investigated the potential mechanisms involved in the inhibitory effect of extracellular ATP on the growth of NPC cells and found that extracellular ATP could reduce Bcl-2 and p-AKT levels while elevating Bax and cleaved caspase-3 levels in NPC cells. Decreased levels of p65 and osteopontin were also detected in the ATP-treated NPC cells. We demonstrated that extracellular ATP inhibited the growth of NPC cells via p65 and osteopontin and verified that P2Y2 overexpression elevated the inhibitory effect of extracellular ATP on the proliferation of NPC cells. Moreover, a dual luciferase reporter assay showed that the level of osteopontin transcription was inhibited by extracellular ATP and P2Y2. ATP decreased the binding of p65 to potential sites in the OPN promoter region in NPC cells.
This study indicated that extracellular ATP inhibited the growth of NPC cells via P2Y2, p65 and OPN. ATP could be a promising agent serving as an adjuvant in the treatment of NPC.
Accumulating evidence has associated purinergic signals, which are induced by extracellular nucleotides, to the processes of several diseases. Extracellular nucleotides, particularly ATP, are important transmitters that mediate various biological effects via purinergic receptors (P2-receptors) in many cell types, and several studies have found that ATP can inhibit tumor growth[3–6]. P2 receptors are subclassified into two main types: P2X and P2Y receptors. P2X receptors are ligand-gated ion channels that are activated by extracellular ATP to elicit a flow of cations, seven of which, P2X1 to P2X7, have been cloned. The metabotropic P2Y receptors belonging to the G-protein-coupled receptor (GPCR) family play important roles in several signaling pathways, and eight P2Y receptors have been cloned and identified as GPCRs in mammals[2, 9]. Although P2Y receptors are distributed in a wide range of normal tissues, P2X receptors are mainly expressed in the nervous system, platelets, and smooth muscle cells (SMCs)[10, 11]. P2Y2 has often been reported to be a functional receptor that transduces several biological signals induced by ATP and UTP, studies largely conducted in normal cells, such as epithelial cells, smooth muscle cells, leukocytes, and nerve cells. It has also been reported that P2Y2 activates nerve growth factor signaling to enhance neuronal differentiation and is also involved in phagocytic clearance[12–15]. However, the role of P2Y2 in tumor cells remains poorly understood, though some reports have described the possible role of P2Y2 in effects of extracellular nucleotides on tumor cells. Osteopontin (OPN) is a secreted arginine-glycine-aspartic acid (RGD)-containing phosphoprotein with a thrombin cleavage site. By binding to several integrins and CD44 variants, OPN plays an important role in tumorigenesis, tumor invasion, tumor growth, and metastasis in many types of cancers[16–18]. OPN has been shown to promote cell survival through the inhibition of apoptosis, and OPN downregulation decreases the motility and invasiveness of tumor cells[19, 20]. Although it has been reported that extracellular nucleotides induce OPN expression in arterial SMCs, their effect on OPN expression in tumor cells has not been examined.
Purinergic signaling has thus far not been investigated in NPC cells, and the effect of extracellular ATP on tumor cell OPN levels is unclear. Therefore, the effects of ATP on NPC cell apoptosis, cell cycle arrest, and cell migration were investigated in the present study, and we also explored whether the effects were caused through P2Y2 and OPN.
Materials and methods
ATP was purchased from Amersco (Solon, Ohio, USA), prepared in water, and stored in aliquots of an appropriate volume at -20°C until use. The antibodies used were anti-OPN (Sigma, USA), anti-P2Y2 (Santa Cruz, USA), and anti-cleaved caspase-3 (Cell Signal Technology, USA); other antibodies were purchased from Beyotime (Nantong, China).
Cell culture and transfection
The cell lines 5-8 F and CNE-2 were gifts from Dr. Shan Jiang, State Key Laboratory of Oncology in Southern China, SunYat-Sen University, Guangzhou, P. R. China. The cells were cultured in DMEM supplemented with heat-inactivated fetal calf serum and penicillin (100 U/ml)/streptomycin (100 mg/mL) in 5% CO2 at 37°C. The transfection of plasmids and siRNA was performed using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions.
The cell lines were seeded in 96-well plates at a density of 5000 cells per well in a volume of 150 μL of culture medium per well. After 24 h, ATP was added to the wells at different concentrations in triplicate. The plates were incubated at 37°C in 5% CO2 for 24 or 48 h. If a transfection was performed, ATP was added to the wells after the transfection for 24 h. A 20-μL sample of MTT solution (5 g/L, dissolved in PBS) was added to each well, and the plates were incubated at 37°C for an additional 4 h. The supernatant was discarded, and l50 μL of DMSO was added to dissolve the formazan product. The absorbance values at 570 nm (A570) were determined using a multi-well plate reader (Tecan, Maennedorf, Switzerland).
Measurement of intracellular calcium
The change of intracellular Ca2+ concentration in the cultured 5-8 F and CNE2 cells was measured using the fluorescence probe fluo-3 with acetoxymethyl ester. The two cell lines were grown in 96-well plates (black well, clear bottom; Greiner Bio One) until confluence. The dyes were loaded into the cells by adding 5 μM fluo-3 AM from 1 mM stock in DMSO to a bath solution containing the following: 20 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM, K2HPO4, 1 mM MgCl2 and 5 mM glucose with the pH adjusted to 7.4. The cells were incubated for 40 min at 37°C in the dark. After loading, the cells were subsequently washed three times in bathing solution, and then, 100 μl of bathing solution was added to each well. The plates were then placed in a multifunctional multi-well plate reader (Genios Plus, Tecan). The fluorescence density was measured after the addition of 100 μl of control buffer or 100 μl of the indicated concentration of ATP in bath solution with or without receptor antagonists (PPADS, suramin). The absorption wavelength was 535 nm, and the excitation wavelength was 485 nm. The fluorescence of intracellular Ca2+ was also observed under inverted fluorescence microscopy.
siRNA sequences and plasmid construction
The coding sequences of P2Y2 and OPN were amplified from CNE-2 cells, and each was cloned into pcDNA3.1. The siRNA used for human OPN knockdown was a mixture of 5′-GUGGGAAGGACAGUUAUGATT-3′ and 5′-GUCUCACCAUUCUGAUGAATT-3′. The siRNA control sequence was 5′-ACGCATGCATGCTTGCTTT-3′. The plasmid used for p65 knockdown was a gift from Dr Yang Zhang (Department of Biochemistry and Molecular Biology, Zhongshan School of Medicine, Sun Yat-Sen University, China) and was based on pSilence2.0-U6, with the following sequence: 5′-CGCTGCAGTTTGATGATGAATTCAAGAGATTCATCATCAAACTGCAGCTTTTTTG-3′. P2Y2-shRNA expression plasmids were constructed based on plasmid pcDNA3.1(+) (Invitrogen, USA) according to a previous report. EGFP was amplified by PCR from pEGFP-N1 (Clontech, USA), and the human U6 promoter was obtained from the human genome. The EGFP fragment was inserted between the KpnI and Bam HI sites of pcDNA3.1(+), and the U6 promoter was inserted into the Bam HI and Eco RI sites of pcDNA3.1(+). The siRNA sequence targeting the human P2Y2 transcript and the control sequence were as follows:
Control (NC), 5′-AATTCTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTT-3′;
Flow cytometry analysis
A cell cycle analysis was performed by flow cytometry. DNA labeling was performed using the Cycletest Plus DNA Reagent kit (BD Biosciences Pharmingen, USA), and the samples were analyzed using a flow cytometer (Beckman Counter, USA). For the detection of apoptotic cells, labeling tests involving both propidium iodide (PI) and annexin-V were performed using an annexin-V staining kit (Invitrogen, USA) according to the manufacturer’s instructions.
Cell migration assays were performed using the Transwell (Costar, USA) system, which allows cells to migrate through 8-μm pore polycarbonate membranes. ATP (100 μM) was included or omitted in the medium in both the upper and lower compartments of the chambers. The chambers were incubated for 24 h at 37°C in humidified 5% CO2 air. The membranes were then washed with PBS, and the cells were fixed with cold methanol for 15 min and stained with crystal violet. The cells beneath the membrane were counted in 5 high-power microscopic fields. Each experiment was performed using three Transwell chambers and repeated three times.
Cells were treated with ATP for 36 h, washed with PBS, and fixed for 20 min at room temperature with 4% paraformaldehyde. The cells were washed again with PBS and permeabilized with Triton X-100 for 10 min at room temperature; the cells were then washed with PBS, incubated with 3% BSA to block non-specific binding sites, and incubated with primary antibodies at 4°C for 15 h. The cells were washed three times with PBS, incubated with the secondary antibody for 1 h at room temperature, and observed using an inverted fluorescence microscope after washing three times with PBS.
Western blot analysis
Cells were exposed to various experimental conditions for the indicated times before being harvested and lysed for protein extraction. The protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad, P. R. China). The blots were visualized using an enhanced chemiluminescence detection system (Amersham, Pittsburgh, USA). β-actin was used as a loading control.
Luciferase reporter assay
5-8 F and CNE-2 cells were seeded at 5 × 103 per well in 96-well plates the day before transfection. A 683-bp promoter region (-365 to +318) of OPN was inserted into the pGL3-Basic luciferase reporter vector (Promega, USA). The cells were co-transfected with 0.1 μg of firefly luciferase reporter construct, 0.01 μg of pRL-TK Renilla luciferase reporter plasmid (Promega, USA), and the pcDNA3.1-P2Y2 vector using Lipofectamine 2000 (Invitrogen, USA). The luciferase activity was examined using a dual-luciferase reporter assay system (Promega, USA) according to the manufacturer’s instructions, and the signal was normalized to the internal Renilla control for the transfection efficiency.
5-8 F and CNE-2 cells treated with or without ATP were utilized for chromatin immunoprecipitation using the EZ ChIP kit (Millipore, USA) according to the manufacturer’s instructions. After elution and purification, the recovered immunoprecipitated DNA samples were used for PCR with primers 5′-CAGTTGCAGCCTTCTCAGC-3′ (forward) and 5′-CCTTTGTTCCACAGGAGACC-3′ (reverse) to amplify a 201-bp segment of the OPN promoter containing the potential p65 binding sites. The PCR products were analyzed by agarose gel electrophoresis.
A statistical analysis was performed using the unpaired Student’s t-test. P values < 0.05 were considered statistically significant.
Effects of extracellular ATP on the proliferation and migration of NPC cell lines
ATP induces apoptosis and S-phase arrest in NPC cells
Intracellular calcium levels are changed in response to ATP
Effects of extracellular ATP on the levels of p65 and OPN in NPC cells
Vector-based RNAi decreases P2Y2 expression
Effects of P2Y2 and OPN on CNE-2 cell chemosensitivity to ATP
To examine the role of P2Y2 and OPN in the effect of ATP on the proliferation of CNE-2 cells, the cells were transfected with constructed plasmids or siRNA for 24 h and then cultured with ATP (100 μM) for another 48 h. An MTT assay was used to evaluate cell viability after treatment with ATP. As shown in Figure 5B, we found that OPN downregulation increased the sensitivity of CNE-2 cells to ATP, whereas OPN overexpression resulted in a higher cell viability compared to the corresponding control group. OPN overexpression itself showed no obvious effect on the viability of CNE-2 cells. We further found that the upregulation of P2Y2 could decrease CNE-2 cell viability, with P2Y2 downregulation having no obvious influence on CNE-2 cell viability. These results indicated that P2Y2 and OPN participate in the effect of ATP on the proliferation of CNE-2 cells.
Effects of P2Y2 expression on CNE-2 cell cycle and apoptosis
The cell cycle distribution was analyzed by flow cytometry after the cells were transfected with the P2Y2 expression vector for 24 h and treated with 100 μM ATP for another 48 h. As shown in Figure 5C, the proportion of pcDNA3.1-P2Y2-transfected cells in G2/M phase decreased (9.3% to 6.8%), whereas those in G1 phase increased (54.7% to 60%) compared to the control group. To assess apoptosis, cells were transfected with the P2Y2 expression vector for 24 h and were then treated with 100 μM ATP for another 48 h before analysis by flow cytometry. The results indicated that overexpression of P2Y2 increased the apoptosis of CNE-2 cells (Figure 5C).
Effect of P2Y2 on the expression of p65 and OPN in NPC cells
P-AKT is involved with OPN in the chemosensitivity of CNE-2 cells to ATP
Our results show that extracellular ATP decreased the viability and inhibited the migration of 5-8 F and CNE-2 cells. Notably, ATP-induced cell cycle arrest in S phase was significant in these NPC cells. Given that certain chemotherapeutic drugs are more cytotoxic to cells arrested in S phase[27, 28], the recruitment of cancer cells to S phase by ATP might sensitize poorly differentiated NPC cells to these drugs. Apoptosis was also induced in the NPC cells treated with ATP. P2 receptors might be involved in these effects. Because P2Y2 receptor is the main subtype for ATP, P2Y2 might participate in the effects of ATP on NPC cells. In the present study, P2Y2 promoted the growth inhibition effects of ATP on NPC cells, similar to the situation in some other tumor cells. However, some reports have shown that ATP can stimulate the proliferation of cancer cells[29, 30], though the underlying mechanism is unclear. For the first time in NPC cells, our study showed that extracellular ATP inhibits the proliferation and migration of NPC cells via the downregulation of p65 and OPN. A recent report provided evidence that ATP could inhibit the migration of NPC cells (CNE2Z) through the blockage of volume-activated chloride channels, which is opposite to the situation in SMCs. OPN, which was detected in both 5-8 F and CNE-2 cells by western blotting, has been studied extensively in several tumor models and was found to be involved in the regulation of several signal transduction pathways and factors, including the AKT pathway[24–26, 32]. One recent report also provided evidence that OPN could regulate the growth of NPC cells. Here, we report for the first time that both OPN and ATP can affect the level of p-AKT in NPC cells, and our results also indicate that the AKT pathway might be involved in the effects of ATP and OPN on NPC cells. As the inhibition of OPN expression can result in a decrease in the metastatic potential of tumor cells, these findings may therefore be of great benefit for patient prognosis. Indeed, a higher level of OPN was detected in the serum of NPC patients, and a higher level of OPN was shown to decrease the sensitivity of NPC cells to radiotherapy[33, 34]. Given that radiotherapy is the main therapy for NPC, the downregulation of OPN by ATP might sensitize poorly differentiated NPC cells to radiotherapy and decrease the metastatic potential of these cells. Clearly, the effect of ATP on OPN expression in other tumor cells needs to be studied further.
Similar to many other purinergic receptors, the function of P2Y2 in tumor cells remains poorly understood. Some reports have proposed a possible function of P2Y2 in tumor cells via changes in ion flux, and new functional purinergic receptors are constantly being reported. In our study, we constructed plasmids to specifically regulate the P2Y2 level and then investigated its potential function. We showed that P2Y2 is involved in the effect of ATP on NPC cells via p65 and OPN and verified that it exhibited growth inhibitory effects on tumor cells on its own, results that are apparently contradictory to the reports suggesting that P2Y2 is involved in the promotion of proliferation[29, 30]. ATP and its receptor P2Y2 might exert opposite effects on migration in different cell types: ATP and P2Y2 were found to stimulate the migration of corneal epithelial cells but inhibit human keratinocyte spread and migration[35, 36]. In the present study, ATP exerted an inhibitory effect on NPC cell motility, whereas P2Y2 itself showed no influence on motility. We also used RT-PCR was used to detect the expression of purinergic receptors in NPC cells, and future studies will be aimed at investigating the function of these receptors in tumor cells. The expression of P2Y2 receptors in 5-8 F and CNE-2 cells was also examined by immunofluorescence microscopy.
It is well known that p65 signaling has important roles in carcinogenesis, cancer development and progression. In addition, p65 signaling was involved in the effects of chemotherapeutic agents, and it might be the attractive targets for the development of new anti-cancer drugs. The relationship between purinergic signaling and p65 signaling in cancer cells is still unclear. One recent report indicated that ATP could inhibit the proliferation of endothelial progenitor cells via inhibiting TLR4 activation induced phosphorylation of p65. In this study, we provided preliminary data about the effects of ATP and P2Y2 on p65 signaling in NPC cells.
In conclusion, the present results show that extracellular ATP inhibits the growth and migration of NPC cell lines, and some of these effects are mediated by the downregulation of p65 and OPN via P2Y2. Therefore, ATP could be a promising agent serving as an adjuvant in the treatment of NPC. P2Y2 and OPN might be potential targets of gene therapy, though further research in NPC cells is needed.
This work was supported by the Provincial & Ministry of education Research Project of Guangdong (Grant number 2012B091100458 to Fujun Zhang), Major project of science and technology of Guangzhou (Grant number 132400027 to Fujun Zhang), and national natural science foundation of China (Grant number 81301769 to Shenghong Zhang).
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