Background
There are well-known side effects of chemotherapy and radiotherapy, mainly due to the toxicity-related impaired function of vital organs [
1,
2]. However, in addition, these therapies induce the unwanted expression and release of several pro-metastatic factors that create a pro-metastatic microenvironment [
1,
3,
4]. Surprisingly, this issue has not been fully investigated thus far.
We propose the novel concept that toxic damage in various organs leads to an upregulation of the expression and activity of several factors in “bystander” tissues, including extracellular nucleotides (EXNs), which provide chemotactic signals to cancer cells that survived the initial treatment. We propose that this mechanism plays an important role in the metastasis of cancer cells and indicates the need to develop efficient anti-metastatic drugs that work in combination with, or follow, standard therapies in order to prevent the possibility of therapy-induced spread of tumor cells [
1,
3‐
5].
Nucleotides leaking through the membranes of damaged cells or released through specific pathways include purine nucleotides and nucleosides (e.g., ATP, ADP, AMP, adenosine) as well as pyrimidine nucleotides (e.g., UTP, UDP), which signal through purinergic receptors expressed in almost every tissue [
6]. There are both nucleoside- and nucleotide-activated receptors, which belong to different receptor families and are distinguishable by their pharmacological properties. While P1 receptors, which are divided into A
1, A
2A, A
2B, and A
3 subtypes, respond to adenosine and its analogues, P2 receptors are activated by ATP and/or other nucleotides. P2 receptors are further subdivided into ionotropic (P2X) and metabotropic (P2Y) receptors, based on structural characteristics [
7,
8]. Ionotropic P2X receptors are assembled in a trimeric form as homo- or heteromers consisting of the subunits designated P2X1–7. P2X receptor channels are activated by ATP, which opens the channel to allow the influx of Ca
2+, Na
+, and K
+. The mammalian metabotropic P2 receptor family contains eight different subtypes, denoted P2Y1, 2, 4, 6, 11, 12, 13, and 14 [
7,
8].
Nucleotides may also be released from cells in response to certain stimulatory agents and affect the cell in an autocrine/paracrine manner. For example, the migration of leucocytes in response to analpylatoxin C5a is potentiated by the release of ATP at the leading edge of migrating cells [
9]. The availability and lifetime of released ATP in a controlled manner for autocrine or paracrine stimulation of purinergic receptors is controlled by a highly efficient enzymatic cascade, including processing that degrades nucleotides (e.g. ATP, ADP, and AMP), finally yielding nucleosides (e.g. adenosine) and thereby regulating activity levels of the various P2 and P1 receptors [
10]. It has been reported that, while the interstitial ATP in normal tissues attains a concentration of up to 1000 nM, the intratumoral ATP concentration can be as much as 10
3–10
4 fold higher [
11].
Taking into consideration the possibility that EXNs affect the behavior of LC cells, we became interested in their role in progression of this tumor. We observed that EXNs accumulate in several murine organs in response to radiochemotherapy and that most of the functional P2X, P2Y, and P1 receptor subtypes are expressed in human LC cells. EXNs were found to modulate the pro-metastatic behavior of LC cells, and their metastasis could be inhibited in immunodeficient mice in the presence of specific small molecule inhibitors of purinergic receptors. Based on these findings, it is clear that EXNs are novel pro-metastatic factors and that inhibition of their pro-metastatic effects via purinergic signaling could become an important part of anti-metastatic treatment.
Methods
Cell lines
We used several human lung cancer cell lines (obtained from the American Type Culture Collection, Manassas, VA), including both non-small cell lung cancer (NSCLC; A549, HTB177, HTB183, and CRL5803) and small cell lung cancer (SCLC; CRL2062 and CRL5853) cell lines. NSCLC cells were cultured in Roswell Park Memorial Institute (RPMI) medium 1640, containing 10 % fetal bovine serum (FBS), 100 U/ml penicillin, and 10 μg/ml streptomycin. CRL2062 cells were maintained in Waymouth’s MB 752/1 medium containing 10 % FBS, 100 U/ml penicillin, and 10 μg/ml streptomycin. CRL5853 cells were cultured in DMEM:F12 medium supplemented with 5 % FBS, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 30 nM sodium selenite (ITS, Lonza, Allendale, NJ), 10 nM hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 10 nM beta-estradiol (Sigma-Aldrich), 4 mM L-glutamine, 100 U/ml penicillin, and 10 μg/ml streptomycin. All cells were cultured in a humidified atmosphere of 5 % CO2 at 37 °C, and the media were changed every 48 h.
Preparation of conditioned media
Pathogen-free C57BL6 mice were purchased from the National Cancer Institute (Frederick, MD, USA), allowed to adapt for at least 2 weeks, and used for experiments at age 7–8 weeks. Animal studies were approved by the Animal Care and Use Committee of the University of Louisville (Louisville, KY, USA). Mice were irradiated with 250, 500, 1000, or 1500 cGy. Twenty-four hours later, bone marrow and plasma were isolated. Conditioned medium (CM) was obtained by 1-h incubation of BM in RPMI at 37 °C. After centrifuging, the supernatant was used for further experiments. In studies with the chemotherapeutic agent vincristine, mice were injected intraperitoneally with 0.9 % NaCl with (0.5 mg/kg or 2 mg/kg) or without vincristine. Twenty-four hours later, organs were isolated, and CM was prepared as described above.
Chemotaxis assay
Chemotaxis assays were performed in a modified Boyden’s chamber with 8-μm polycarbonate membrane inserts (Costar Transwell; Corning Costar, Lowell, MA, USA) as described previously [
3,
4]. In brief, cells detached with 0.25 % trypsin were made quiescent by incubation for 1–3 h in appropriate medium (RPMI, DMEM-F12, or Waymouth’s MB 752/1), supplemented with 0.5 % (NSCLC) or 0.2 % (SCLC) bovine serum albumin (BSA). The cells were then seeded into the upper chamber of an insert (pretreated with 1 % gelatin) at a density of 3.5 × 10
4 (NSCLC) or 10 × 10
4 (SCLC) in 110 μl. The lower chamber was filled with pre-warmed medium containing test reagents. All nucleotides (adenosine triphosphate, ATP; adenosine diphosphate, ADP; adenosine monophosphate, AMP; adenosine; uridine triphosphate, UTP; guanosine triphosphate, GTP; thymidine triphosphate, TTP; cytidine triphosphate, CTP) were obtained from Sigma-Aldrich. Medium supplemented with BSA was used as a negative control. In some experiments, cells were pretreated with the P2 receptors inhibitor
iso-PPADS (Tocris, Minneapolis, MN), the A
1 receptor agonist PSB63 (Tocris), the A
2A receptor antagonist ANR94 (Tocris), the A
2B receptor antagonist PSB603 (Tocris), the A
3 receptor antagonist MRS3777 (Tocris) or the stimulator ivermectin (Sigma-Aldrich) for 15 min at 37 °C. Inhibitors were also added to the lower chambers and were present throughout the experiment. In experiment with apyrase, apyrase (Sigma) was added to lower chamber together with HGF. After 24 h, the inserts were removed from the Transwell supports. The cells that had not migrated were scraped off with cotton wool from the upper membrane, and the cells that had transmigrated to the lower side of the membrane were fixed and stained with HEMA 3 (manufacturer’s protocol, Fisher Scientific, Pittsburgh, PA) and counted on the lower side of the membrane using an inverted microscope.
Adhesion assay to fibronectin
Cells were made quiescent for 3 h with appropriate medium containing BSA and incubated with nucleotides for 10 min. Subsequently, cell suspensions (5 × 103/100 μL) were added directly to 96-well plates coated with fibronectin and incubated for 5 min at 37 °C. The wells were previously coated with fibronectin (10 μg/ml) overnight at 4 °C and blocked with 0.5 % BSA for 1 h before the experiment. Following incubation, the plates were vigorously washed three times to remove non-adherent cells, and the number of adherent cells was counted using an inverted microscope.
Real-time quantitative reverse-transcription PCR
Total RNA was isolated from LC cells with the RNeasy kit (Qiagen, Valencia, CA). Human lung RNA was obtained from Ambion (Austin, TX). The RNA was reverse transcribed with MultiScribe reverse transcriptase, oligo(dT), and random-hexamer primer mix (Life Techonologies, Foster City, CA). Quantitative assessment of mRNA levels was done by real-time reverse transcription PCR (qRT-PCR) on an ABI 7500 Fast instrument with Power SYBR Green PCR Master Mix reagent. Real-time conditions were as follows: 95 °C (15 s), 40 cycles at 95 °C (15 s), and 60 °C (1 min). According to melting point analysis, only one PCR product was amplified under these conditions. The relative quantity of a target, normalized to the endogenous β2-microglobulin gene as control and relative to a calibrator (normal lung tissue), is expressed as 2
−ΔΔCt (fold difference), where Ct is the threshold cycle, ΔCt = (Ct of target genes) − (Ct of the endogenous control gene, β2-microglobulin), and ΔΔCt = (ΔCt of lung cancer cell line sample cDNA for target gene) − (ΔCt of normal lung tissue cDNA for the target gene). All primers that were used for qRT-PCR are listed in Additional file
1: Table S1.
Flow cytometry
For measuring A2B expression cells were detached using non-enzymatic reagent (CellStripper, Corning), followed by 2 h incubation in appropriate medium with 0.5 % BSA. Next cells were washed with PBS, fixed by 15 min incubation at 4 °C in BD Cytofix/Cytoperm solution (BD Biosciences, Franklin Lakes, NJ, USA), washed again and incubated for 30 min in 0.5 % BSA in PBS. Cells were stained with primary rabbit polyclonal anti-A2B antibody (1:25, Bioss Inc, Woburn, MA, USA) for 1 h at 37 °C. Than cells were washed and incubated with secondary antibody Alexa Fluor 488 goat anti-rabbit (1:100, Life Technologies). Cells were then analyzed using the LSR cell cytometer (BD Biosciences). For all other receptors cells were detached and mechanically dissociated to a single cell suspension using TrypLE™ Express (Life Technologies) for 1 min at room temperature, passed through a 40 μm cell strainer and then washed twice with phosphate-buffered saline (PBS). Cells were subsequently fixed with 4 % (PBS) for 30 min at RT, washed and incubated for 30 min in a blocking solution containing 0.05 % Triton X-100, 0,05 % Tween-20 and 5 % FBS in PBS. Staining with primary antibodies was performed after 2 h incubation with primary antibodies: rabbit polyclonal anti-P2X4 (1:200, Santa Cruz Biotech, Dallas, TX, USA), rabbit polyclonal anti-P2X7 (1:200, Aviva Systems Biology, Corp., San Diego, CA, USA), goat polyclonal anti-P2Y1 (1:200, Santa Cruz Biotech), rabbit polyclonal anti-P2Y12 (1:500, Alomone, Jerusalem, Israel). Cells then were washed and incubated for 40 min with Alexa Fluor 488 donkey anti-rabbit (1:1,000, Life Technologies) or Alexa Fluor 488 donkey anti-goat (1:1,000, Life Technologies) secondary antibodies. Cells were analyzed with the AttuneVR cytometer (Life Technologies). The analysis of the data was performed using the FlowJo 7.2.5 or 7.6.3 software (FLOWJO, Ashland, OR, USA). Unstained cells and cells incubated with isotype control were used as controls.
Mean relative of fluorescence intensity analysis is presented as a value of mean of fluorescence intensity for stained cells divided by mean of fluorescence intensity obtained for control cells.
Cell proliferation
Cells were seeded in culture flasks at an initial density of 1.25 × 104 cells/cm2 (NSCLC) or 6 × 104 cells/cm2 (SCLC). After 24 h, the medium was changed to new medium supplemented with 0.5 % BSA, and the cells were cultured in the presence or absence of nucleotides. Full medium (with FBS) was treated as a positive control. The cell number was calculated at 24, 48, and 72 h after the change of medium. At the indicated time points, cells were harvested from the culture plates by trypsinization and counted using Trypan Blue and a Neubauer chamber.
Phosphorylation of intracellular pathway proteins
The HTB177 and CRL5803 cell lines were kept overnight or 6 h, respectively, in medium containing 0.5 % BSA to render the cells quiescent. The cells were then stimulated with nucleotides at 37 °C for 5 min, then lysed for 20 min on ice in RIPA lysis buffer containing protease and phosphatase inhibitors (Santa Cruz Biotech, Santa Cruz, CA). The extracted proteins were separated on a 12 % SDS-PAGE gel and transferred to a PVDF membrane. Phosphorylation of the serine/threonine kinase AKT (phospho-AKT473) and p44/42 mitogen-activated kinase (phospho-p44/42 MAPK) was detected by rabbit and mouse antibodies (Cell Signaling, Danvers, MA, USA), respectively, with HRP-conjugated goat anti-rabbit and anti-mouse secondary antibodies (Santa Cruz Biotech). Equal loading in the lanes was evaluated by stripping the blots and reprobing with anti-p42/44 MAPK monoclonal antibody (clone no. 9102, Cell Signaling) and anti-AKT polyclonal antibody (Cell Signaling). The membranes were developed with enhanced chemiluminescence (ECL) reagent (Amersham Life Sciences, Arlington Heights, IL), dried, and subsequently exposed to film (Hyperfilm, Amersham Life Sciences).
Calcium measurements by microfluorimetry
Cell suspensions (5 × 10
4/100 μL) were seeded onto a black 96-well, clear-bottom plate in appropriate medium and cultured until reaching 80–90 % confluence. The intracellular calcium concentration transient was measured using the FlexStation Calcium 4 Assay Kit (Molecular Devices Corp.), as reported elsewhere [
12]. To explore the mechanism of agonist efficacy, the indirect fluorescence were determined using a FlexStation III plate reader (Molecular Devices Corp., Sunny Valley, CA). Briefly, the cells were incubated for 1 h at 37 °C with the calcium indicator solution containing 2.5 mM probenecid in a 200-μL final volume per well. The fluophore was excited at 485 nm, and the emitted fluorescence was detected at 525 nm. Changes in free intracellular calcium concentration ([Ca
2+]
i
) were determined by subtracting the minimum fluorescence intensity from the maximum fluorescence intensity (Fmax–Fmin), normalized by the baseline resting state.
Quantitation of ATP, UTP and adenosine
The ATP levels secreted by cultured LC cells were measured using the Adenosine 5′-triphosphate Bioluminescent Somatic Cell assay kit (Sigma-Aldrich), according to the manufacturer’s instructions. Briefly, cells were seeded into black 96-well microplates with transparent bottoms, and the light emitted by luciferase activity was detected using a FlexStation III plate reader (Molecular Devices Corp., Sunny Valley, CA), with the light intensity proportional to ATP concentration. The ATP level in bone marrow (BM), conditioned medium from BM, and plasma were measured using the ATP Colorimetric/Fluorometric Assay Kit and Deproteinizing Sample Preparation Kit (BioVision, Milpitas, CA, USA), according to the manufacturer protocol. Fluorescence analysis was performed with Ex/Em set at 535/585 nm. Adenosine level was measured using Adenosine Fluorymetic Assay Kit (BioVision) and UTP level was measured using UTP ELISA Kit (MyBio Source, San Diego, CA, USA) according to the manufacturers’ protocols.
Bone marrow cell lysates and conditioned media were obtained by flushing the bone marrow tibia and femur cavities and resuspending cells in 3 ml of RPMI medium. Cells suspensions were than centrifuged (680 × g, 10 min, 4 °C) and supernatants were collected and employed in experiments as conditioned media. Bone marrow cells were than washed with PBS, counted using Tuerk solution and cells pellets were fast frozen and stored in −80 °C. To obtained cell lysates frozen pellets were resuspended in 200 μl of PBS cells and subjected to ultrasonication 10 times (1 s) followed by centrifugation at 20,000 × g for 10 min, 4 °C to remove cell debris.
Transplant of LC cells into immunodeficient mice
To study the effects of the pharmacological inhibition of P2X or A2b signaling on the metastasis of lung cancer in vivo, HTB177 cells were pretreated with iso-PPADS (100 μM), PSB603 (1 μM), or vehicle alone for 1 h. The cells were then washed and injected intravenously (2.5 × 106 per mouse) into severe combined immunodeficient (SCID)-Beige inbred mice (five mice per group) that were either untreated (control) or previously irradiated with 1000 cGy for 24 h. Marrows, livers, and lungs were removed 48 h after injection of these cells, and the presence of LC cells (i.e., murine–human chimerism) was evaluated as the difference in the level of human α-satellite DNA expression. DNA was amplified in the extracts isolated from BM-, liver-, and lung-derived cells using real-time PCR. Briefly, DNA was isolated using the QIAamp DNA kit (Qiagen). Detection of human satellite and murine β-actin DNA levels was conducted using real-time PCR and an ABI Prism 7500 Sequence Detection System. A 25-μl reaction mixture containing 12.5 μl SYBR Green PCR Master Mix, 300 ng DNA template, 5′-ACC ACT CTG TGT CCT TCG TTC G-3′ forward and 5′-ACT GCG CTC TCA AAA GGA GTG T-3′ reverse primers for α-satellite DNA, and 5′-TTC AAT TCC AAC ACT GTC CTG TCT -3′ forward and 5′- CTG TGG AGT GAC TAA ATG GAA ACC -3′ reverse primers for β-actin DNA was used. The Ct value was determined as before. The number of human cells present in the murine organs (the degree of chimerism) was calculated from the standard curve obtained by mixing different numbers of human cells with a constant number of murine cells.
Statistical Analysis
Statistical analysis of the data was done using T–test (for data with normal distribution) or Whitney–Mann test (data without normal distribution) with p < 0.05 considered significant or one way ANOVA with Bonferroni post hoc p ≤ 0.05 (calcium measurements and analysis of ATP in culture medium).
Discussion
One of the most challenging clinical problems is the tumor recurrence and metastasis of cancer cells that survive standard treatment [
1,
13,
24]. To explain these phenomena, we have proposed that one of the unwanted side effects of radiochemotherapy is the induction of a pro-metastatic microenvironment in normal tissues that are damaged by the treatment, due to an increase in certain peptide- and lipid-based chemottractants [
1,
3,
4,
13]. In parallel, we have hypothesized that “leaky” cells damaged by radiochemotherapy also release nucleotides that, as demonstrated in the past, are potent chemotactic factors for both normal [
9,
25] and malignant cells [
16,
18].
In support of a role of purinergic signaling in cancerogenesis, it is well known that malignant tumors promote strong inflammatory reactions together with necrosis, and nucleotides may leak from damaged cells into the extracellular medium or even be released by specific pathways as part of tumor cell metabolism and anti-tumor protection mechanisms [
26‐
29]. What is also important, nucleotides may be released from the damaged cells in response to radiochemotherapy, as shown in this paper. In fact, we found an increase in ATP level in irradiated murine tissues, including BM and liver, which are known sites for cancer metastasis. It has also been reported that nucleotides may be released from cells stimulated by the fifth complement cleavage fragment, the anaphylotoxin C5a [
9], and it is well known that the complement cascade is activated in cancer patients [
30,
31]. In our studies, we found that HGF may also increase the secretion of ATP from LC cells, and HGF, along with C5a, is upregulated in response to radiochemotherapy [
13,
32]. The question remains whether, in addition to C5a and HGF, other factors that are released in tissues in response to anti-cancer treatment such as certain chemokines [
13] or bioactive lipids [
3,
4] also increase the release of nucleotides from target cells, but this requires further studies.
In our work, we focused mainly on the biological effects of ATP, ADP, AMP, and adenosine, which are well-established ligands for G-protein coupled P1 and P2Y receptors and ligand-gated ion channel P2X receptors [
23]. While P1 receptors are activated by adenosine and A
1 receptor subtypes also by AMP [
33], P2X receptors are activated by ATP, and P2Y receptors respond to ATP, ADP and UTP [
34]. In our studies, we demonstrated that all of these nucleotides stimulate human LC cells. We also found responsiveness of LC cells to TTP, CTP, and GTP. Despite some suggestions that these nucleotides may also stimulate some P2X receptors, we cannot exclude that observed effect is due to receptor independent cell stimulation. As support of such hypothesis, similar phenomenons were already described for ATP [
11] and adenosine [
35]. However, we focused our current work on the most relevant EXNs and nucleoside, which are ATP, ADP, AMP, and adenosine.
We learned that human NSCLC and SCLC cells express several functional purinergic receptors. Stimulation by EXNs promoted migration and adhesion of LC cells. These functional responses of LC to nucleotides are supported by the activation of intracellular pathways, including MAPKp42/44 and AKT phosphorylation, as well as [Ca2+]
i
transients.
Nucleotides have already been reported to stimulate proliferation of some malignant cells, including colon adenocarcinoma and melanoma cells [
36,
37]. To our surprise, however, we found that, if added to LC cell cultures, nucleotides did not stimulate their growth. On the other hand, we detected ATP in conditioned media harvested from LC cells, and inhibition of purinergic signaling in these cells by
iso-PPADS and caffeine negatively affected their proliferation. This suggests the involvement of autocrine signaling axes in the proliferation of LC cell lines. In support of a role for autocrine purinergic signaling in regulating the biology of LC cells, autocrine signaling via release of ATP and activation of the P2X7 receptor was found by another group to enhance the motility of human LC cells [
17].
Nevertheless, data on the effect of nucleotides and nucleosides on the proliferation of LC cells are somewhat controversial. For example, it has been reported that treatment of A549 cells with adenosine results in their senescence, both
in vitro and
in vivo, through induction of cell cycle arrest and senescence in a p53/p21-dependent manner [
38]. A similar effect has been observed after exposure of the PC14 lung adenocarcinoma cell line to ATP [
39]. However, in another report, ATP stimulation of P2Y receptors increased the proliferation of human lung epithelial tumor cells [
21]. These differences may be explained by the much higher concentrations of ATP employed in those studies compared with the concentrations that we used in our work.
It is well known that EXNs may affect different aspects of LC biology. For example, ATP was found to sensitize LC cells to cisplatin-induced apoptosis [
40] and enhance the antitumor effect of etoposide in PC14 and A549 LC cells [
39]. Moreover, it has been reported that extracellular ATP may be internalized by cancer cells by micropinocytosis, which induces an increase in intracellular ATP and drug resistance [
11]. It was also shown that ATP- or UTP-mediated activation of P2Y2 induced cancer cell invasion through increased production of VEGF by cancer cells [
41] and that adenosine receptors have been found to regulate VEGF expression under hypoxic environment in different tissues [
42]. On the other hand, EXNs are potent chemoattractants for mesenchymal stromal cells and thus may attract these cells and promote stromalization of the growing tumor [
43]. Similarly, EXNs may exert an effect on endothelial progenitors and thereby promote tumor vascularization [
44]. Altogether, given these data, purine and pyrimidine nucleotides can be considered crucial orchestrators of both directly and indirectly regulated pro-metastatic potential of tumor cells.
What is most important in our report is that, by employing pro-metastatic assays
in vitro and
in vivo, we have demonstrated for the first time that purinergic signaling may be an attractive target for small molecule antagonists of purinergic receptors to inhibit the metastatic spread of LC cells. Our results employing receptor antagonists lend further support to this concept. On the other hand, it is known that degradation of EXNs in the extracellular space is regulated by enzymatic cascades, including ectonucleoside triphosphate diphosphohydrolases (E-NTPDase 1, also known as CD39; E-NTPDases 2, 3, and 8), ectonucleotide pyrophosphatases/phosphodiesterases (E-NPPs), ecto-alkaline phosphatases, and ecto-5′-nucleotidase (also known as CD73), which degrade nucleotides (e.g. ATP, ADP, and AMP), finally yielding nucleosides (e.g. adenosine) and thereby regulating activity levels of the various P2 and P1 receptors [
45‐
47]. In addition to purinergic receptors, these enzymes are potential targets for small molecule inhibitors to control migration and metastasis of LC cells. This is of particular importance, since, as mentioned above, the concentration of EXNs in tumor tissue could be very high [
11,
48]. However, the evidence supporting a functional role of ectonucleotidases in purinergic signaling varies considerably between enzyme species and thus should be taking into consideration when looking for possible anti-cancer targets [
46,
47].
We are aware that the results that we generated with established human LC cell lines need to be verified with LC patient primary cells. It will be important to establish whether the pattern of purinergic receptor expression has prognostic value and whether it correlates with more malignant and metastatic phenotypes.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
All authors participated in the design, interpretation of the studies and analysis of the data and review of the manuscript; GS, TG, CL, AA-I, and ZS conducted the experiments, MZR, GS, MM and HU wrote the manuscript, GS and TG prepared Figures, MZR provided funds.