Molecular and cellular pharmacologyFeasibility study of B16 melanoma therapy using oxidized ATP to target purinergic receptor P2X7
Introduction
Adenosine 5′-triphosphate (ATP) is released into the extracellular space in response to stress stimuli, such as shear stress, stretching, hypoxia, inflammation, osmotic swelling, and cell death (Yegutkin, 2008). ATP interacts with and activates P2 receptors in an autocrine/paracrine manner. Though the mechanism of ATP release is not yet fully understood, there have been several studies of stress-induced ATP release. The mechanism is dependent on cell type and the nature of the stress stimulus, and is mediated by various pathways, including a maxi-anion channel (Sabirov et al., 2001), a volume-sensitive outwardly rectifying chloride channel (Hisadome et al., 2002) and P2X7 receptor channel (Pellegatti et al., 2005). The concentration of ATP in peri-plasmalemmal space reaches the micromolar range, which is sufficient to activate P2 receptors (Yegutkin, 2008). The activation regulates many physiological functions, such as apoptotic and/or necrotic cell death (Di Virgilio et al., 1998, Hillman et al., 2003, Burnstock, 2006, Tsukimoto et al., 2005, Tsukimoto et al., 2006, Tsukimoto et al., 2007), production of pro-inflammatory cytokines (Takenouchi et al., 2008, Takenouchi et al., 2009), and shedding of CD62L (Scheuplein et al., 2009). As P2X7 receptor, it has already been well established that the P2X7 receptor mediates cell death in lymphocytes treated with a high concentration of ATP (Hillman et al., 2003, Tsukimoto et al., 2005, Tsukimoto et al., 2006, Burnstock, 2006). In addition, activation of P2X7 receptor induces an increase of cation permeability, followed by plasma membrane depolarization. But, although P2X7 receptor mediates ATP release, the mechanism involved remains unclear.
Expression of P2X7 receptor has been reported in several types of cancers (Per et al., 2002, Greig et al., 2003, Calvert et al., 2004, Raffaghello et al., 2006, Solini et al., 2008). Increased expression was also found in B16 melanoma cells, and it was proposed that increase of P2X7 receptor might be a biomarker of cancer (Slater et al., 2003, White et al., 2005, Deli et al., 2007). ATP is known to be secreted by cancer cells into their microenvironment and could be responsible for P2X7 activation. However, it is not yet clear whether this activation directly contributes to the malignant progression of cancer.
The microenvironment of solid tumors is a heterogeneous, complex milieu for tumor growth and survival. In particular, hypoxia and acidosis are common features of solid tumors (Wike-Hooley et al., 1984, Helmlinger et al., 1997, Brown and Giaccia, 1998, Sutherland, 1998, Stubbs et al., 1999). The resulting low-pH conditions might lead to increased ATP release and activation of P2X7 receptor, which in turn might enhance cancer progression (Künzli et al., 2011, Ryu et al., 2011).
We already have found that gamma-radiation evokes ATP release from melanoma cells, and that P2X7 receptor channel plays a significant role in mediating this ATP release. Based on these results, we considered that it might be feasible to treat melanoma by blocking ATP release and P2X7 receptor signaling with a selective P2X7 antagonist, such as oxidized ATP (oxATP). Here, we show that oxATP inhibits B16 melanoma growth, both in vitro and in vivo.
Section snippets
Materials
Dulbecco's modified Eagle's medium (DMEM), RPMI1640 medium (phenol red free), penicillin, streptomycin, dithiothreitol (DTT), and L-glutamine were purchased from Wako Pure Chemical Industries (Osaka, Japan). ATP, oxATP, GdCl3, arachidonic acid, EtBr, glibenclamide (GC), 1,2,3,4,5,6-hexachlorocyclohexane (lindane), arachidonic acid (AA) and flufenamic acid (FFA) were purchased from Sigma-Aldrich (St. Louis, MO). Selective inhibitors of P2X7 receptor,
Low pH induces ATP release from B16 melanoma cells; identification of the release pathway
We examined ATP release from B16 melanoma cells for 10 min post-stimulation with HBSS buffer adjusted to pH 7.4, 7.0, 6.8, or 6.4 by means of luciferin–luciferase assay. As shown in Fig. 1A, the largest ATP release was observed at pH 6.4. Therefore, the time course of ATP release was examined at the fixed pH of 6.4. As shown in Fig. 1B, the concentration increased soon after the low-pH stimulation, reached a peak at 10 min, and then decreased to almost the basal level within 30 min.
In the
Discussion
Though it is well known that various stresses induce ATP release from cells, there has been no previous report of low-pH-induced ATP release and activation of P2 receptors.
In this study, we first established that low pH induces ATP release. We found that the concentration of ATP in culture medium of low-pH-stimulated B16 melanoma cells was elevated soon after the stimulation, reached a peak at 10 min, and then fell to almost the basal level within 30 min. Intracellular ATP is released through
Conclusion
In summary, we have shown for the first time that a low-pH environment induces ATP release and P2X7 receptor activation in cancer cells, suggesting that extracellular ATP acts as a novel intercellular signaling molecule. Our results indicate that P2X7 receptor antagonists are promising candidates for the treatment of solid cancers.
Acknowledgment
This work was supported in part by a Grant-in Aid for Scientific Research ‘KAKENHI’ for Young Scientists (B) (no. 19790097) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to M.T.), in part by a Sasagawa Scientific Research Grant from The Japan Science Society (to M.T.), and in part by grant for Center for Technologies against Cancer (CTC) from Tokyo University of Science (to M.T.).
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Effect of P2X7 receptor on tumorigenesis and its pharmacological properties
2020, Biomedicine and PharmacotherapyCitation Excerpt :Further use of P2X7R antagonists (A438079 and AZ10606120) could reduce the levels of caspase-3, caspase-1, p21 and Cdc25c, and inhibit the growth of pancreatic cancer [171]. A large number of related studies have shown the usefulness of P2X7R-related antagonists and inhibitors (such as AG1478, A438079, OXATP, and Quinolinone derivatives) to reduce the expression of P2X7R in cancer cells and inhibit the growth and migration of tumor cells [172–174]. Through the mentioned research reports, it is believed that the use of P2X7 receptor-related antagonists and inhibitors can reduce P2X7R over-expression in tumor cells, the activity of, and the growth, proliferation, migration, and invasion of tumor cells.
A Purinergic Trail for Metastases
2017, Trends in Pharmacological SciencesCitation Excerpt :In these cases, ATP functions as an ‘alarm signal’ alerting and activating in an autocrine/paracrine manner the surrounding immune cells to fight microbes and/or initiate tissue reparative responses [13–15] or to localize and engulf apoptotic cells [16]. Many stimuli are endowed with the ability to induce the release of ATP and other nucleotides from normal cells [17–19], but this is also true for tumor cells [20–23]. Once in the extracellular milieu, ATP (and ADP) is quickly hydrolyzed to AMP (by the ectonucleoside triphosphate diphosphohydrolase, NTPDase/CD39) and subsequently to ADO (by the ecto-5′-nucleotidase also indicated as CD73) [24–27].
Pharmacological targeting of ion channels for cancer therapy: In vivo evidences
2016, Biochimica et Biophysica Acta - Molecular Cell ResearchCitation Excerpt :In the same zebrafish model, in vivo invasiveness of the P2X7R-positive MDA-MB-435 cells was reduced by 50% using 10 μM emodin. Two other studies independently demonstrated that oxidized ATP (0.5 mg/mouse 3 times i.p.), a selective antagonist of P2X7, significantly reduced tumor volume in a B16 melanoma-bearing mice model [111] as well as in a colon cancer CT26 model (reduction by 90% with 3 intratumoral injections of oxATP (600 mmol/L) [112]. The same authors also showed that intratumoral injection of 300 nmol/L of the selective P2X7 inhibitor AZ10606120 caused a strong inhibition of melanoma B16 tumor growth as well.
P2X7 Receptor as a Therapeutic Target
2016, Advances in Protein Chemistry and Structural BiologyCitation Excerpt :Transforming growth factor beta is a component of tumor microenvironment that affects cancer cell migration and was shown to influence vesicular release of ATP and activation of P2X7, causing cytoskeletal rearrangements leading to cancer cell movement (Takai, Tsukimoto, Harada, & Kojima, 2014; Takai et al., 2012). Of interest, also this phenomenon is dependent upon variations in intracellular Ca2 + and PI3K activation (Hattori et al., 2012). P2X7 triggered Ca2 + rise was known for long to result in cell proliferation (Adinolfi et al., 2005) also causing activation of the Ca2 +-sensitive nuclear factor NFATc1 (Adinolfi et al., 2009, 2010).
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These authors are contributed equally to this work, and share the first authorship.