Gliomas are the most common primary tumors of the central nervous system (Wesseling and Capper, 2018). These types of brain tumors have particularly aggressive behavior with a high recurrence rate. Although the current therapeutic approach combines surgical intervention, irradiation, and adjuvant chemotherapy, the prognosis is still very poor for these tumors. Thus, there is a need to find new strategies to improve glioma treatment and reduce its recurrence rate. Adenosine is a key mediator of several biological functions involving multiple signaling pathways (Borea et al., 2018) and mainly operates through four G-protein coupled receptors named A₁, A_2A, A_2B, and A₃. Adenosine A₁ and A₃ receptors are coupled to Gi/o-proteins and inhibit adenylyl cyclase activity. In turn, adenosine A_2A and A_2B receptors are coupled to Gs-proteins and stimulate adenylyl cyclase activity. Adenosine can be formed intracellularly and exported via transporters or extracellularly with the participation of CD73 activity as an adenosine-generating enzyme from adenine nucleotides, as ATP, released from cells. Adenosine can be transformed into inosine by adenosine deaminase activity (Fredholm et al., 2011). Because adenosine is considered one of the major constituents within the tumor microenvironment (Di Virgilio and Adinolfi, 2017), adenosinergic signaling has emerged as a potential therapeutic strategy in cancer (Allard et al., 2012). However, the precise procedure to target adenosine-mediated signaling remains under discussion as two different tissues may be affected, the tumoral cells and the immune system. Adenosine seems to be involved in tumor generation, growth, invasion, angiogenesis, and metastasis through activation of all four adenosine receptor subtypes (Gessi et al., 2011; Allard et al., 2012). Nevertheless, adenosine appears to have an opposite biological action as tumor-derived adenosine induces A_2A receptor activation from immune cells, leading to an immunosuppressive state of the immune system and, thus, facilitating tumor growth (Ohta et al., 2006). In addition, CD73 is suggested as a key enzyme in tumor growth (Zhang, 2012; Yan et al., 2019). In agreement, its inhibition or depletion causes cell growth inhibition in different in vitro (Bavaresco et al., 2008; Zhu et al., 2017) and in vivo models (Stagg et al., 2011, 2012). In fact, several drugs and antibodies targeting CD73 are under study in clinical trials due to their potential role in cancer (Buisseret et al., 2018).

Resveratrol (RSV) is a polyphenolic compound present in plants, such as peanuts and grapes, and it shows multiple healthy properties in several diseases, including cancer (Carter et al., 2014; Jiang et al., 2017; Ko et al., 2017). This phytochemical emerged as a promising molecule since the first time its effectiveness was reported against cancer in both in vitro and in vivo models (Jang et al., 1997; Kiskova et al., 2020). Unfortunately, the action mechanism by which this polyphenol exerts its antitumoral activity remains not well understood. Recently, we reported that RSV acts as a non-selective agonist for adenosine receptors in rat C6 glioma cells (Sanchez-Melgar et al., 2019). Moreover, RSV induces in vivo changes in adenosinergic signaling by modulating the functionality of A₁ and A_2A receptors in the brain from SAMP8 mice after long-term RSV supplementation in their diet (Sanchez-Melgar et al., 2018). Therefore, the aim of the present work was to investigate whether RSV treatment is able to modulate adenosine-converting enzymes and whether adenosinergic signaling is somehow involved in the antitumoral action of this polyphenol in C6 glioma cells.

Results presented herein indicate that exposure of C6 glioma cells to RSV caused cell growth inhibition in a time- and concentration-dependent manner by accumulating cells in the G₁ phase. Caspase-3 activity was increased after treatment. Furthermore, adenosine-converting enzyme activities (i.e., CD73 and ADA) were significantly reduced in RSV-treated cells. In agreement, increased levels of extracellular adenosine were detected, whereas intracellular adenosine remained unaltered. Interestingly, adenosine A₁ and A₃ receptors seem to contribute in part to the antiproliferative effect of RSV because the blockade of these receptors partially ameliorates the effect of RSV.

Resveratrol is attracting attention in the prevention of several diseases, including cancer (Jang et al., 1997). However, the precise molecular mechanisms behind its antiproliferative action remain to be clarified. Our results show that RSV induced reduction in cell viability in a time- and concentration-dependent manner and a higher activity of caspase-3, an early apoptosis marker. The absence of apoptotic bodies in the nuclei and a cell cycle arrest in the G₁ phase, together with a lower percentage of cells in the S and G₂/M phases, suggests an antiproliferative effect of RSV through cell cycle arrest. A limitation of the cell cycle analysis we performed is that only a single parameter was used for DNA content analysis, and it would be interesting to use an additional specific marker to distinguish between the cell cycle phase. Anyway, we found an IC₅₀ value of 78.0 μM (95% CI: 33.2–167.4) for 24 h RSV treatment, very similar to the 85.26 ± 2.14 μM previously reported in C6 cells by Zielinska-Przyjemska et al. (2017). However, these authors report a cell cycle arrest in the S phase after 100 μM RSV exposure for 24 h. Cycle arrest in the S phase is also reported after 100 μM RSV exposure for 48 h (Wang et al., 2015) or 210 μM RSV for 24 h (Zhang et al., 2007). In agreement with our results, it is reported that 100 μM RSV induced cell cycle arrest in the G₁ phase in breast cancer cells after 24 h but not 48 h treatment (Medina-Aguilar et al., 2016) and in human melanoma cells (Wu et al., 2015) after 48 h. Thus, RSV inhibition of cell cycle progression seems to be cell line specific.

Purinergic signaling is involved in cancer cell proliferation (Di Virgilio and Adinolfi, 2017), and both P2 (Di Virgilio et al., 2018) and P1 (Allard D. et al., 2017) receptors as well as CD39 and CD73 enzymes (Allard B. et al., 2017) could be new targets in cancer. The role of adenosine signaling in cancer is still under debate although it is well accepted that adenosine can promote cancer cell proliferation in several tumors through its receptors (Ohta, 2016; Kazemi et al., 2018). It is reported that adenosine A_2B receptors (Sepulveda et al., 2016) and CD73 (Zhang, 2012) are overexpressed in many cancer types. Moreover, adenosine has been found at higher levels in the tumor microenvironment when compared with normal tissue (Ohta et al., 2006) even at a range of 50–100 μM (Vaupel and Mayer, 2016). This tumor-derived adenosine seems to promote cancer cell growth in a receptor-dependent manner as reviewed elsewhere (Ohta, 2016), but it also facilitates immune escape by activating A_2A receptors in T cells (Ohta et al., 2006), suggesting a protumor effect of adenosine. However, other authors report a cytotoxic action of adenosine in human cervical cancer cells (Mello Pde et al., 2014), indicating that adenosine might exert a differential action depending on the type of cancer. This cytotoxic effect was also observed in our study when C6 cells were treated with 100 μM adenosine for 24 h. In contrast, it is reported that 100 μM adenosine increased cell proliferation by 36% in U138MG glioma cells (Bavaresco et al., 2008), which might indicate that adenosine action depends not only on the type of cancer (e.g., glioma) but also on the cancer cell line.

Despite the well-known antitumor effect of A_2A receptor depletion or pharmacologic inhibition by enhancing the antitumor immune response in mice (Ohta et al., 2006), the specific role of adenosine receptors in the tumor itself remains under debate (Gessi et al., 2011; Di Virgilio and Adinolfi, 2017; Borea et al., 2018; Gorain et al., 2019). Adenosine receptors could display an important action on cancer cell growth, invasion, angiogenesis, and even metastasis (Ohta, 2016; Kazemi et al., 2018). Our work reveals that prolonged pharmacologic blockade of A_2A receptors with ZM241385 results in a discrete but significant reduction in the cell viability in conjunction with higher caspase-3 activity, suggesting an antiproliferative effect in cancer cells for A_2A receptor antagonists. Other authors report similar data in lung adenocarcinoma tumor cells (Mediavilla-Varela et al., 2013). In a previous study, our group discovered that RSV binds and acts as a non-selective adenosine receptor agonist in C6 glioma cells and that acute RSV treatment altered the A_2A receptor/Gs-protein coupling, leading to the inhibition of the cAMP generation upon pharmacologic stimulation of the A_2A receptor with CGS21680. Moreover, adenylyl cyclase (AC), PKA protein levels, and basal AC activity were significantly increased after 100 μM RSV treatment for 24 h (Sanchez-Melgar et al., 2019). This dramatic alteration of A_2A receptor signaling after RSV treatment makes it difficult to analyze the possible contribution of these receptors to the antiproliferative effect of RSV at least by combining RSV with agonists or antagonists for A_2A receptors as employed here. Diet supplementation with RSV also caused the desensitization of A_2A receptors in the brain from SAMP8 mice (Sanchez-Melgar et al., 2018). These results might support that the alteration of A_2A receptor signaling could be involved in the antiproliferative action of RSV. It is reported that elevation of intracellular cAMP levels through either activation of AC or inhibition of PDEs leads to PKA activation and positively correlates with reduced cell proliferation of C6 glioma cells. Interestingly, the elevation of cAMP levels with forskolin induces cell cycle arrest of C6 glioma cells in the G₂/M phase. In comparison, inhibition of PDEs not only inhibits cell growth via the cAMP/PKA cascade, but also triggers cell death through caspase-3/-7 activation (Safitri et al., 2020). It is described that anticancer agents, such as RSV, may act by modulating cell cycle–associated proteins, such as cyclins, cyclin-dependent kinase (CDK), and CDK inhibitors (Wolter et al., 2001). CDK inhibitors are shown to be the downstream targets of caspase-3 activation, and loss of these inhibitors can result in the aberrant upregulation of CDKs that have been associated with apoptotic cell death (Jin et al., 2000). Therefore, RSV-induced G₁-phase cell cycle arrest could be mediated through the caspase/cyclin-CDK pathways. In agreement, the protein content of the cycle arrest proteins CDK2, CDK4, cyclin D1, PCNA, and P21 is reported to be decreased in a concentration-dependent manner in RSV-treated (100 μM, 24 h) HCT116 and Caco-2 cells compared with control cells (Liu et al., 2014). Moreover, RSV inhibits human U251 glioma cell proliferation and induces G₀/G₁ growth arrest, and these effects are reduced by the CDK inhibitor olomoucine (Jiang et al., 2005). These RSV effects on cell cycle and viability via caspase-3 activation could be modulated by different adenosine receptors after their activation by binding of adenosine or even RSV. In fact, treatment of C6 glioma cells with 25 μM Cl-IB-MECA reduced Bcl-2 expression and increased caspase-3 activity after 24 h of treatment. This apoptotic effect was observed only with activation of the A₃ receptor, whereas activation of the A₁ or A_2A receptors did not induce significant apoptotic effects (Appel et al., 2001). However, activation of A₁ receptors with CPA increased the cell viability and reduced apoptosis, and the antagonist DPCPX significantly induced apoptosis and caspase-3 expression in MCF-7 cells (Dastjerdi et al., 2016).

Among the molecular targets of RSV reported to date, the AMP-activated protein kinase (AMPK) can be found (Kulkarni and Canto, 2015). The activation of AMPK is reported to suppress the proliferation of various cancers via the regulation of cell cycle progression, apoptosis, autophagy, inhibition of protein synthesis, and de novo fatty acid synthesis. AMPK causes G₁ cell cycle arrest via upregulation of the tumor suppressor protein p53, which upregulates p21, a CDK inhibitor (Motoshima et al., 2006). AMPK is a trimer with α-, β-, and γ-subunits. The α-subunit contains the kinase domain, and its Thr¹⁷² residue is phosphorylated (p-AMPK) by an upstream kinase and determines its regulation. The binding of AMP and, to a lesser extent, ADP to the γ-subunit stimulates AMPK activity. Thus, changes in the ATP/ADP or ATP/AMP ratio lead to the allosteric activation of AMPK (Herzig and Shaw, 2018). Therefore, enzymes involved in the modulation of AMP levels due to the conversion of AMP to adenosine (i.e., 5′NT) or adenosine to AMP (i.e., adenosine kinase) could control AMPK activation. Extracellular adenosine activates AMPK (Aymerich et al., 2006); however, PKA-mediated inhibition of AMPK via increased inhibitory phosphorylation of AMPK^Ser173 and reduced activating phosphorylation of AMPK^Thr172 is reported (Djouder et al., 2010; Aw et al., 2014). This PKA-mediated inhibition of AMPK could take place in C6 glioma cells as cAMP/PKA signaling is significantly increased, and the AMPK activation (measured as the p-AMPK/AMPK ratio) is not modified in these cells after 100 μM RSV treatment for 24 h (Sanchez-Melgar et al., 2019). Therefore, the cell cycle arrest elicited by RSV in C6 glioma cells seems to be independent of the AMPK/p53/p21 mediated inhibition of CDKs.

The enzyme CD73 has gained attention since it was discovered that adenosine is one of the major constituents in the tumor microenvironment (Di Virgilio and Adinolfi, 2017) and that this tumor-derived adenosine facilitates the immune escape by activating the A_2A receptor in T and NK cells (Ohta and Sitkovsky, 2014). CD73 overexpression in tumor cells is associated with the pathogenesis (Yan et al., 2019), progression (Yu et al., 2018), and poor prognosis in several types of cancers, including HNSCC (Ren et al., 2016a), triple-negative breast cancer (Loi et al., 2013), oral squamous cell carcinoma (Ren et al., 2016b), and high-grade serous ovarian cancer (Turcotte et al., 2015), among others. Therefore, targeting CD73 with selective inhibitors or antibodies is being considered as a promising therapeutic strategy against cancer (Stagg et al., 2010). Our data indicate a significant reduction in the CD73 activity located in the plasma membrane fraction after RSV exposure, suggesting that RSV might affect adenosine production in the pericellular space. Nevertheless, a lower enzymatic activity of ADA was also found after RSV treatment, leading to lower deamination of adenosine into inosine and significantly decreased inosine levels. A limitation of the technique employed here to measure CD73 activity could be that we cannot rule out the contribution of alkaline phosphatase in the conversion of AMP to adenosine (Zimmermann, 2021). However, the presence of 100 μM levamisole, a selective alkaline phosphatase inhibitor, during the assay determining CD73 activity in the human cerebral cortex modified the activity of CD73 in neither membranes nor cytosolic fraction by using the same assay conditions as here (Alonso-Andres et al., 2018). This reduced CD73 activity could promote an increased level of ATP. It is well known that extracellular ATP may exhibit a cytotoxic effect in cancer cells depending on the concentration (Vultaggio-Poma et al., 2020). Among P2 receptors, the P2 × 7 receptor subtype seems to be the main player in ATP-dependent biological actions. Prolonged activation of P2 × 7, via high levels of extracellular ATP over an extended time period, can lead to the formation of a macropore, leading to depolarization of the plasma membrane and, ultimately, to cell death (Lara et al., 2020). However, our results show that, after 24 h of P2X stimulation with BzATP, C6 cell viability did not change, and this prolonged stimulation was unable to modify the RSV effect.

A highly active CD73 enzyme has been detected in glioblastoma (Ludwig et al., 1999) and glioma (Bavaresco et al., 2008). Independently of its enzymatic role, CD73 can mediate cell–cell adhesion being a coreceptor in T cell activation or regulate cell interaction with ECM components and migration on them. Acting as a docking molecule, CD73 mediates migration and invasion of A375 melanoma cells (Sadej and Skladanowski, 2012) and glioblastoma invasiveness (Fenoglio et al., 1997) through focal adhesion kinase activation. Interestingly, RSV was able to regulate the invasion of cancer cells by modulating such focal adhesion kinase (Buhrmann et al., 2017), which could be mediated by the RSV effect on the CD73 enzyme.

The precise molecular mechanism by which RSV modulates CD73 activity is not clarified yet. It is described a possible link between hypoxia-inducible factor-1 alpha (HIF-1α) and CD73 (Sotnikov and Louis, 2010). Hypoxic conditions in the tumor trigger HIF-1α activation and, in turn, an upregulation of the CD73-adenosine pathway (Li et al., 2017), which is able to promote tumor growth and metastasis (Zhang, 2012). RSV treatment reduced HIF-1α in cancer cells in vitro (Zhang et al., 2005), which might explain the reduction in the CD73 activity observed in RSV-treated cells in our study. Of interest, moderate hypoxia (24 h at 5% O₂) produces increased endogenous adenosine levels in C6 glioma cells and the downregulation and upregulation of A₁ and A_2A receptors, respectively. However, HIF-1α was not modulated by moderate hypoxia, and C6 cells were resistant to cell death elicited by hypoxic insult (Castillo et al., 2008).

A growing body of evidence indicates that dual blockade of CD73 and A_2A receptors could enhance the antitumor response (Beavis et al., 2015; Young et al., 2016). In this sense, RSV induces the reduction in CD73 activity as observed in our study and changes the A_2A receptor downstream signaling from activation to inhibition of adenylyl cyclase as we reported previously (Sanchez-Melgar et al., 2019).

Reduced CD73 and ADA activities as reported herein result in increased extracellular levels of adenosine after RSV exposure. However, RSV-induced cell growth inhibition seems to be independent of the activation of adenosine receptors by extracellular adenosine because of (1) an adenosine concentration of 50 μM, similar to that achieved after RSV treatment, and 75 μM were unable to reduce cell viability, (2) 100 μM adenosine treatment slightly decreased cell viability but to a lesser extent than observed with RSV treatment, and (3) ADA presence during RSV treatment did not impede the RSV effect on these cells. Instead, direct activation of adenosine receptors by RSV itself acting as a non-selective agonist seems to be involved as we recently suggested (Sanchez-Melgar et al., 2019). Pharmacological inhibition of the A₁ receptor with DPCPX or the A₃ receptor with MRS1220 during RSV treatment resulted in a significantly reduced RSV effect, suggesting possible participation of reduced levels of cAMP after RSV binding to these Gi-protein coupled receptors (i.e., A₁ and A₃). Moreover, prolonged pharmacologic inhibition of the A_2A receptor with ZM241385 or the A_2B receptor with PSB1115 partially mimicked the RSV-induced effect on C6 cell viability. As mentioned, RSV treatment altered the A_2A receptor/Gs-protein coupling, leading to the inhibition of the cAMP generation upon pharmacologic stimulation of the A_2A receptor with CGS21680 after RSV treatment (Sanchez-Melgar et al., 2019). All these data might indicate that the reduction of C6 cell growth upon RSV treatment could be related to the inhibition of cAMP levels through adenosine receptor modulation, mainly A₁ and A₃ receptors.

In summary, our study suggests that a reduced CD73 activity located in the plasma membrane in addition to a fine-tuned modulatory role of adenosine receptors could be involved, at least in part, in the antiproliferative action of RSV in C6 glioma cells (Figure 10).

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

MM and JA: conceptualization, writing—review and editing. AS-M, MM, and JA: formal analysis. MM: funding acquisition. AS-M and SM-L: investigation. AS-M and MM: writing—original draft. All authors have read and agreed to the published version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.