Future Perspectives

### **Chapter 7**

Immunomodulatory Effects of a M2-Conditioned Medium (PRS® CK STORM): Theory on the Possible Complex Mechanism of Action through Anti-Inflammatory Modulation of the TLR System and the Purinergic System

*Juan Pedro Lapuente*

#### **Abstract**

Co-culture of primary or mesenchymal stem cells (MSC) with M2 macrophages produces a very special conditioned medium with a recognizable and stable cytokine pattern (PRS CK STORM), independent of the donor, with unique anti-inflammatory properties. This product can regulate certain pathways of inflammation in an antiinflammatory manner, including TLR3, TLR4, the inflammasome, and the purinergic system. The anti-inflammatory action of PRS CK STORM is demonstrated both by its composition and by its action in *in vitro* and *in vivo* inflammatory models. The study of the mechanism of action showed changes in the pattern of toll-like receptors (TLR) and purinergic receptors, with an increase in the relative expression of mRNA encoding A2a and A3 receptors, together with a decrease in the relative expression of mRNA encoding P2X7 receptors. Second, it mitigated the adverse effects of a systemic inflammatory process in mice, especially in comparison with a known anti-inflammatory drug (Anakinra). Thus, due to its profile in terms of biosafety and efficacy, PRS CK STORM may be a strong candidate to treat inflammatory processes, such as cytokine storm associated with severe infectious processes, including COVID-19.

**Keywords:** co-culture, cytokines, ADP, cross-talk, toll-like receptors (TLRs), macrophages (M), mesenchymal stem cells (MSCs)

#### **1. Introduction**

Inflammation is the response of an organism's immune system to damage caused to its cells and vascularized tissues by bacterial pathogens and by any other biological,

chemical, physical, or mechanical aggressor. Such an inflammatory response must be self-limiting in time and intensity since, if this is not the case and if there is no perfect coordination between the innate and adaptive immune systems, a severe systemic inflammatory syndrome with positive feedback systems will occur, eventually causing a cytokine storm that can lead to multi-organ failure [1, 2]. In the establishment, maintenance and termination of this cytokine storm, at the molecular level, in cases of sepsis and severe viral infections such as that associated with COVID-19, the toll-like receptors (TLRs), NOD-like receptors (NLRs), and RIG-like helicase receptors (RLRs), cytokines, chemokines and growth factors, and the purinergic system will be fundamental in the establishment, maintenance, and termination of this cytokine storm.

In the late 1990s, the ability of infectious agents (bacteria, viruses, zoonoses, or parasitic and/or fungal infections) to trigger cytokine storm syndrome (CSS) was first described with the recognition of a case series of hemophagocytic lymphohistiocytosis (HLH) of viral origin [3]. Such a cytokine storm is basically characterized by an exaggerated production of proinflammatory and profibrotic soluble mediators (especially IL-1β, IL-6, and TNF-α), together with an aberrant immunopathological reaction, involving an uncoordination between the innate and adaptive immunity system, there being generally an overactivation of the innate immune system, the main cellular actors being macrophages, dendritic cells, monocytes, neutrophils, and T lymphocytes [4–7]. As a consequence of this cytokine storm, a situation of multi-organ hyperinflammation will be provoked, which usually affects mainly the lung and pancreas, among other organs, and which usually results in acute respiratory distress syndrome (ARDS) and/or acute lung injury (ALI), which can lead to multi-organ failure.

Although the association of increased levels of proinflammatory and profibrotic cytokines and chemokines with increased levels of morbidity and mortality following an infectious process is well known, we still lack a suitable drug to treat the cytokine storm [8].

The innate immune system is able to recognize molecular structures specific to viruses, bacteria, fungi, and other pathogens; these structures are known as pathogenassociated molecular patterns (PAMPSs) [9–11]. PAMPSs are small-molecule sequences that are repeated in groups of pathogens recognized by the so-called pattern recognition receptors (PRRs). These include the toll-like receptors (TLRs) family of membrane receptors, NOD-like receptors (NLRs) and RIG-like helicase receptors (RLRs), among which the NLRP receptors stand out, oligomeric structures called inflammasomes, responsible for generating the mechanism of pyropoptosis by hyperproduction of hyperinflammatory cytokines, used as a trigger for the hyperproduction of IL-1β and IL-18 [12]. These molecular patterns are essential for the recognition of microorganisms by innate immunity cells, which respond differently depending on the microorganism identified [9–11].

Analyzing the detection capabilities of all these receptors, both DAMPS and PAMPS, we conclude that the main receptors involved in innate immunity against infections are TLR2, TLR3, TLR4, TLR7, TLR9, NOD1, NOD2, RIGI, and NLRP3. In **Table 1**, we summarize the PAMPS and DAMPS that are able to activate them [13–17].

Considering the different receptors involved in cytokine storms associated with infectious processes, we can deduce that the activation of the transcription factors AP-1 (activator protein 1) and NF-kβ (nuclear factor kappa light chain enhancer of activated B cells), both common denominators in almost all pattern recognition pathways, will provoke the dreaded cytokine storm, resulting in a state of generalized *Immunomodulatory Effects of a M2-Conditioned Medium (PRS® CK STORM)… DOI: http://dx.doi.org/10.5772/intechopen.104486*


*TLR 3 receptors are activated mainly by viruses, although SARS COV-2 is not activated to the same extent as other viruses, such as influenza virus, respiratory syncytial virus (RSV), or herpes virus. TLR 4 receptors are activated mainly by bacteria, but activation is also seen in viruses, although it is unclear whether this is due to a posteriori DAMPS. TLR 7 receptors are mainly activated by viruses. TLR 9 receptors are activated by both RNA and DNA so that viruses, bacteria, fungi, or any pathogen can activate them. Cytoplasmic receptors, since they detect most pathogen fragments, including DNA and RNA, can be activated by both viruses and bacteria, although NOD1 is activated more strongly by bacteria. It appears that RIG I is only activated by viral and not bacterial RNA [13–17]. (++ maximal activation, + intermediate or mild activation, no activation).*

#### **Table 1.**

*TLR 2 receptors are activated mainly by bacteria and fungi, and although their activation has been described in SARS COV-2 infection, it is possible that this is due not directly to the SARS COV-2 virus but to the existence of a concomitant bacterial infection.*

hyperinflammation. The most affected organs are lung or pancreas, with the consequent associated fibrotic reaction, producing irreparable anatomopathological damage with loss of function in the most affected organs.

A fact especially associated with the cytokine storm associated with SARS COV-2 is that a decrease in the production of type I interferons is observed, which causes dysregulation in the coordination of the innate and adaptive immunity systems, facilitating the appearance of the dreaded severe pneumonia that on many occasions determines the patient's admission to the ICU [18].

It is very difficult to explain the existence of a cytokine storm by the activation of a single receptor. If this were so, treatment of the cytokine storm by a single monoclonal antibody, for example, a monoclonal antibody against IL-1β or against IL-6, would always be effective, and this we know almost never occurs. Moreover, even if in the first instance only one of the receptors is activated, the simple initiation of its metabolic cascade will provoke the appearance of DAMPS that will stimulate other receptors. If we add to this the fact that in the majority of cytokine storms associated with infections we do not see a single causative pathogen, but rather a group of them, we will understand that there is almost always a joint activation of several of these receptors, producing between them phenomena of agonism and synergy, as well as antagonism [19]. Any of them can have agonistic relationships with others, if they are stimulated at the same time. However, if these same receptors are activated with a significant time lag between them of hours or even days, the most likely mutual relationship they will establish will be one of antagonism [19]. Thus, the types of cytokines and chemokines that will be released as a result of the activation of the different receptors will depend on the sets of receptors that are primarily activated by PAMPS and, once initiated, such release of pro-inflammatory and profibrotic mediators will be prolonged and augmented over time by positive feedback from the same receptors or even the addition of others, by the stimuli elicited by DAMPS, which could lead to reactive phenomena even autoimmunity.

In the cytokine storm, we must also consider the intervention of the purinergic system [20–22]. Extracellular adenosine triphosphate (eATP) or its enzymatic degradation products, such as ADP, AMP, and adenosine, can stimulate a number of membrane receptors [23]. More specifically, the P2X7 receptor is widely distributed on innate cells of the adaptive immune system, a system that constitutes the first line of defense against invading pathogens. These cells are lymphocytes, granulocytes, macrophages (including microglia), and dendritic cells in peripheral tissues [24–26]. Activation of the P2X7 receptor has been associated with the establishment and prolongation of inflammation and cytokine storm in septic infections, including SARS-COV-2 infection [27– 29]. The stimulation of the P2X7 receptor by adenosine triphosphate (ATP) causes the activation of the NLRP3 inflammasome, and consequently of caspase 1, stimulating this the exaggerated secretion of IL-1β and IL-18 [30]. For all these reasons, the ideal immunomodulatory treatment of the cytokine storm associated with moderate and severe infections should include the P2X7 receptor (generating antagonism) or P1-like receptors (generating agonism) as a therapeutic target [29].

The treatments tested to date to control cytokine storms associated with infectious processes have been based on the use of monoclonal antibodies used alone or in combination. The hypothesis put forward by our group proposes as a treatment a biological therapy based on the use of allogeneic-conditioned medium derived from M2-type macrophages and enriched with mesenchymal stem cells (MSCs). Mesenchymal stem cells, placed in co-culture with macrophages, not only respond to macrophages and adjust their secretome accordingly but also induce macrophages to respond to them, creating a feedback loop that contributes to immune regulation [31]. In the complex composition of this conditioned medium are present all growth factors, cytokines, and chemokines that are naturally produced by M2-type macrophages

#### *Immunomodulatory Effects of a M2-Conditioned Medium (PRS® CK STORM)… DOI: http://dx.doi.org/10.5772/intechopen.104486*

and MSCs, associated with innate immunity respecting natural pleiotropic relationships. The immunomodulatory cytokine profile of this medium confers a potent antiinflammatory and anti-fibrotic action, and even, thanks to the results obtained with the secretome of MSCs on macrophages stimulated with TLR7/8 ligand, possibly antiviral [32]. Different studies have shown that the secretome of these two cell types is modified and modulated when co-cultures of these cells are performed [33]. The immunomodulation mechanisms mediated by MSCs are due, among other factors, to the release of PGE-2 (prostaglandin E-2) and TSG-6 (TNF-stimulated gene 6 protein) [34]. M2 (anti-inflammatory) macrophages secrete high levels of IL-10 and low levels of IL-12p70 and IL-17, in a process that is directly mediated by other factors produced by MSCs (such as IL-6 and HGF) [35, 36]. It has been experimentally demonstrated that factors secreted by pro- or anti-inflammatory macrophages activate the immunomodulatory potential of MSCs. In this regard, IL-10 release by anti-inflammatory macrophages activates MSCs to release PGE-2 [37], which in turn modulates macrophages producing a cascade of additive molecular interactions in favor of immunotolerant and anti-inflammatory mechanisms. Basically, the polarization of macrophages to M2 type with immunoregulatory phenotype will be maintained [38], which, in turn, will express more IL-6, IL-10, and IGF-1 and inhibit their production of IL-12 and TNF-α. MSCs are also capable, through secretion of these same factors, of inhibiting the migration, maturation, and differentiation of dendritic cells [39–41]. Similarly, monocyte-derived M2 macrophages co-cultured with MSCs have been shown to increase mitochondrial function and ATP turnover, both in vitro and in vivo, resulting in an increase in the ADP/ATP ratio [42]. In addition, MSCs maintain ATPase and CD73 enzymatic activities on their surface, converting ATP to ADP and AMP to adenosine, respectively [43]. Adenosine, the last molecule in these reactions, has immunoregulatory functions through the activation of the P1 receptor [44]. Importantly, activation of monocyte P1 receptors, such as A2A and A2B, inhibits TNF-α production [44].

Several previous experiences demonstrate how secretomes from both cell types possess immunomodulatory properties. For example, direct injection of the supernatant of cultured mesenchymal stem cells (MSCs) containing a variety of growth factors, prostaglandins, and cytokines can be applied to the treatment of kidney injury [45]. Both co-culture with M2 macrophages and treatment with M2 macrophage supernatant have also been shown to increase endothelial cell viability in a bacterial lipopolysaccharide-generated lung sepsis model [46]. In addition, the efficacy and safety of multiple sclerosis treatment by intravenous infusion of conditioned medium from mesenchymal stem cell culture have also been demonstrated [47].

The advantage of using the complete conditioned medium versus one of its purified components lies in the synergistic mechanism between its different components [48], the result of subjecting the cell populations to a culture that, in vitro, attempts to emulate the anti-inflammatory, anti-fibrotic, and regenerative immunomodulatory microenvironment that occurs in vivo in diseased tissue.

#### **2. Material and methods**

Production and characterization of allogeneic-conditioned medium derived from M2-type macrophages and enriched with MSCs.

First, to obtain MSCs, a lipoaspirate sample was obtained from which the stromal vascular fraction (SVF) was extracted, following the protocol described by Lapuente

et al. [49]. SVF was harvested by centrifugation under the same conditions as earliermentioned, seeded at a density of approximately 30,000 cells per cm2 in 100-mm diameter culture plates (this and all culture plastic used was from Corning, Corning, NY, USA) and cultured at 37°C and 5% CO2 in culture medium (DMEM + 10% fetal bovine serum (FBS) + 1% P/S). At 24 h, the culture was washed with phosphatebuffered saline (PBS) to remove nonadherent cells and the adherent cell population, called processed lipoaspirate (PLA), was cultured to subconfluence under the same conditions as earlier-mentioned, changing the culture medium three times a week and performing the necessary passages with trypsin 0.05% (Gibco), until a homogeneous population of mesenchymal-type stromal cells, also called mesenchymal stem cells (MSCs), was obtained. After culture, the cells were frozen at a freezing ramp of 0.5°C/ min to 80°C in freezing medium composed of 10% dimethyl sulfoxide (DMSO, Sigma) in FBS or culture medium, then immersed in liquid N2 and maintained until use.

Secondly, monocytes were isolated from one altruistic blood donation bag of 450 ml with 12% citrate–phosphate-dextrose (Grifols, Barcelona, Spain) from the blood bank of the Fuenlabrada Universitary Hospital. To isolate the leukocytes, each bag was divided into 50-ml tubes (Corning) and centrifuged at 1500 x g for 10 min at room temperature (RT). The intermediate band, leukocyte buffy coat, was collected and deposited on a clean tube. Immediately, 24 ml of this concentrate was carefully placed on 18 ml of Ficoll Histopaque 1077 (Sigma) and centrifuged at 400 g for 30 min at room temperature (RT) and without brake. The mononuclear cell band was collected, and after adding PBS in a 1:1 ratio, centrifuged at 300 x g for 5 min at RT. The supernatant was discarded and the resulting pellet was resuspended in a fivefold volume of erythrocyte lysis buffer and incubated at RT for 10 min. Subsequently, a 10-fold volume of PBS was added and centrifuged under the same conditions as earlier-mentioned to obtain the cell pellet after discarding the supernatant. This last wash was repeated once more and, after this, the resulting peripheral blood mononuclear cell pellet (PBMC) was resuspended in CTS-AIM-V medium (Gibco) supplemented with 0.1% Dipeptiven 200 mg/ml (Frenesius Kabi Austria GmbH, Graz, Austria) and cultured in T-175 culture flasks (Corning, approximately 200 million PBMCs per flask) at 37°C and 5% CO2 atmosphere for 90 min. The next step was to wash the flasks twice with plenty of PBS to remove unattached cells. The cells were immediately lifted with a cell scraper (Corning) to obtain a cell suspension in PBS, which was centrifuged for 5 min at 300 g at room temperature. The resulting pellet was resuspended in AIMV + Dipeptiven + 10 ng/ml M-CSF (R&D Systems, McKinley, Minneapolis, USA) for co-culture. All cell counts and viabilities (trypan blue exclusion method) were performed with an automatic counter TC20 (BioRad, Hercules, CA, USA), strictly following the manufacturer's instructions, marking a lower threshold of 8 μm to disregard possible erythrocytes, platelets, and other contaminating cellular debris.

Third, co-culture was established to produce the conditioned medium. For this purpose, the obtained monocytes were seeded at a density of 500,000 cells/cm2 in inserts (Transwels, with a polyethersulfone membrane of 1 μm pore size, from Corning) of 6-well plates and cultured under standard conditions for 4 days with the described medium. When the culture medium was removed, the inserts were washed twice with PBS and added to the plates on which the MSCs had been seeded and cultured in pass 4 (24 h earlier, in Corning 6-well plates at a density of 10,000 cells/cm2 under standard conditions), previously washed twice with copious PBS and using CTS-AIMV-V medium supplemented with 0.1% Dipeptiven to maintain the co-culture under standard conditions of temperature and CO2 concentration. The co-culture was maintained

#### *Immunomodulatory Effects of a M2-Conditioned Medium (PRS® CK STORM)… DOI: http://dx.doi.org/10.5772/intechopen.104486*

for 4 weeks by collecting the conditioned medium and adding fresh medium twice a week. To preserve the different collections, they were immediately frozen by immersion in carbonic snow and kept at 80°C until analysis. To perform the analysis, the cultures were thawed at 4°C and analyzed immediately after filtering through a 0.45 μm pore nitrocellulose filter (Merck KGaA, Darmstadt, Germany). To obtain secretome controls for MSCs and different monocytes, the different cell populations were cultured separately under the conditions described for co-culture.

Subsequently, phenotypic characterization of MSCs and monocytes was performed and samples were taken from both populations at time 0, 7 days, 14 days, and 28 days. MSCs were lifted with trypsin and monocytes with scraper as described earlier and, after centrifugation at 300 g 5 min at 4°C, resuspended each cell type in PBS, permeabilized the monocytes with Perm/Wash buffer (BD Biosciences, Franklin Lakes, NJ, USA), and incubated the cells for 15 min at RT and in the dark with the following fluorochrome-conjugated antibodies (and their related isotypes as negative controls) at 1:50 concentration: CD73-APC, CD90-APC, CD45-FITC, HLA-DR-FITC, CD31-PE, CD68-FITC, and CD163-PE (all from BD Biosciences). The fluorescence minus one technique was used to adjust the voltages and compensate for fluorescence, and propidium iodide (Sigma) was used to determine dead cells according to the manufacturer's instructions. A Guava EasyCyte flow cytometer (Merck) was used to acquire the samples and InCyte software (Merck) was used to analyze the results.

To quantify the secretome of both cell types and the co-culture, 30 growth factors, cytokines, and chemokines were quantified using either ELISA or Multiplex assay (ProcartaPlex 23 PLEX, Invitrogen, Grand Island, NY, USA), strictly following the manufacturer's instructions. A Luminex Labscan 100 plate reader (Luminex Corporation, Austin, TX, USA) was used for the determinations. The molecules quantified by Multiplex were the following: MIP1-α, IL-2, IL-6, TIMP-1, IL-8, IL-10, IL-12 P70, IL-1 RA, RANTES, GM-CSF, leptin, HGF, MMP-3, MCP1, BNGF, EGF, adiponectin, TNFα, MMP-1, TRAIL, FGF-2, PDGF-BB, and VEGF-A. For quantification of IGF-1, BMP-6, IL-1β, IL-4, TGF-β1, TGF-β3, and VEGF-C, a double sandwich ELISA technique was used following the manufacturer's instructions (DuoSet ELISA kit, R&D) and quantification was determined using an iMark plate reader (BioRad).

#### **2.1 Generation of the** *in vitro* **inflammation model**

THP-1 cell line culture and subsequent differentiation to macrophages were performed to generate in vitro models of biosafety and efficacy. THP-1 monocytic cells (CellLineService, cat. No.: 300356) were cultured and expanded using RPMI 1640 (Lonza, Basile, Switzerland) supplemented with 10% fetal bovine serum (FBS) (Corning, NY, USA), 1% penicillin/streptomycin (P/S) (Lonza), 1 mM sodium pyruvate (Lonza), and 1% MEM nonessential amino acids (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) starting now THP-1 medium. Cells were maintained at a density ranging from 2.5 <sup>10</sup><sup>5</sup> to 10<sup>6</sup> cells/ml to ensure adequate growth and a stable phenotype. Forty-eight hours prior to lipopolysaccharide (LPS) stimuli, cells were differentiated into resting macrophages using phorbol 12-myristate 13-acetate (PMA) (Sigma–Aldrich, Saint Louis, MO, USA) at 5 ng/ml in THP-1 medium as described in the protocol used by Park et al. [50]. After this differentiation process, the cells were used for our experiments. All cell cultures were maintained at 37°C in an atmosphere of 5% CO2 and 98% relative humidity. The in vitro inflammation model was generated by differentiating 400,000 THP-1/ml cells in exponential growth phase into resting

macrophages in 12-well plates (Nunc, ThermoFisher) (final volume 1 ml) and after 48-h pretreatment with PMA, the cells were washed three times with 0.5 ml of tempered THP-1 medium without PMA and allowed to incubate for 30 min before LPS stimuli. Once at rest, rest, the cells were treated with 10 ng/ml LPS (Sigma– Aldrich) in RPMI 1640 medium and the investigational product, which had been previously quenched at room temperature or quenched THP-1 medium, using as control the same THP-1 culture treated with the same amount of LPS, but adding in this case 10 μg/ml hydrocortisone. The final volume of each well was 1 ml with 400,000 cells each. Stimulation was carried out for 5 h.

#### **2.2 Evaluation of the possible mechanism of action of the proposed conditioned medium**

After 5 h of stimulation, supernatants were removed from each well, divided into aliquots, and flash-frozen by immersion in dry ice for further analysis. Total RNA was extracted from the cells using an RNeasy Plus Mini kit (Qiagen, Hilden, Germany), and extraction was carried out strictly according to the manufacturer's instructions. This kit included a genomic DNA removal step. The resulting RNA was eluted from the columns using nuclease-free water, divided into aliquots, and stored at 80°C to avoid degradation by environmental RNAases. From the 40 μl of RNA solution from each sample, an aliquot was extracted to assess RNA integrity and concentration. Total RNA integrity was assessed by agarose gel electrophoretic run of total RNA on a 2% agarose-TBE gel for 30 min at 120 V and 400 mAh. Quantification of total RNA was performed by a fluorimetric method using a highly sensitive fluorimetric kit (Qubit HS RNA quantification kit, Applied Biosystems, ThermoFisher). The cDNA was synthesized from total RNA for quantitative PCR of our genes of interest. A high-capacity cDNA reverse transcription kit (Applied Biosystems) was used for synthesis, and a total of 150-ng total RNA was used, for each synthesis reaction. Each sample had 5 cDNA synthesis reactions to achieve sufficient volume for downstream applications. The synthesis protocol was performed using an RNAase inhibitor following the manufacturers' recommendations, and their protocol was strictly followed. Random hexamers were used to perform reverse transcription of all mRNAs into doublestranded cDNA. After synthesis, the cDNAs were divided into aliquots and stored at 20°C, for later use. An aliquot of these cDNAs was extracted for quantification using a Qubit dsDNA HS Assay kit (Applied Biosystems). Primer concentrations were optimized using a cDNA pool to determine the most appropriate concentrations of the primers in the qPCR protocol. For such determination, a standard PCR was performed using a 2 PCR MasterMix (DreamTaq HotStart PCR MasterMix) (Applied Biosystems). Cycling conditions were 98°C for 3 min, then 35 cycles at 95°C for 45 s, 60°C for 30 s, and 72°C for 30 s. After these 35 cycles, the temperature was set at 72°C for 5 min and then held at 4°C indefinitely. The optimal primer concentration was determined by selecting the sharpest specific bands on agarose electrophoresis, uncontaminated by the presence of primer dimers at the front of the gel or nonspecific products. Ideal primer concentrations were 250 nM for forward and reverse primers. Primer sequences and amplicon sizes are attached in **Table 2**.

The following Thermo Fisher primers (coupled to FAM) were also used for A2a (Hs00169123\_m1), A3 (Hs04194761\_s1), and P2X7 (Hs00175721\_m1) receptors.

Subsequently to perform qPCR, total RNA was extracted from 400,000 THP-1 cells using a Qiagen RNeasy plus mini kit (Qiagen, Hilden, Germany). THP-1 cells had been previously differentiated to resting macrophages using 5 ng/ml phorbol 12*Immunomodulatory Effects of a M2-Conditioned Medium (PRS® CK STORM)… DOI: http://dx.doi.org/10.5772/intechopen.104486*


#### **Table 2.**

*Primer sequences and amplicon sizes.*

mystirate 13-acetate (PMA) (Sigma–Aldrich, Saint Louis, MA, USA) in cell culture medium 48 h prior to the experimental model. A total of 0.75 μg of RNA was retrotranscribed to cDNA using a high-capacity cDNA synthesis kit (ThermoFisherScientific, Waltham, USA) employing random hexamers. For qPCR, 10 ng of cDNA per reaction was amplified using a Power SYBR-Green PCR master mix (Applied Biosystems, Thermo Fisher) on a StepOnePlus real-time PCR machine (Perkin Elmer, Waltham, MA, USA) and using the primers listed in **Table 1** (primer table). Thermal cycling conditions included an initial denaturation step at 95°C for 5 min, followed by 40 cycles at 95°C for 30 s, 60°C for 30s, and 72°C for 30s. Melting curve analysis of each qPCR was performed on the final products. Messenger RNA fold changes were calculated using the ΔΔΔCt method with GAPDH as a calibrator gene.

Finally, in order to have an approximation of the mechanism of action of our PRS® CK STORM conditioned medium, two studies were performed. Firstly, a quantification of the ATP/ADP ratio contained in the drug. The Sigma–Aldrich colorimetric ADP/ATP ratio assay kit (Ref: MAK135) was used for this purpose, and the Biorad iMark plate reader was used for its reading. Secondly, a quantification of extracellular ATP in THP-1 cells placed in culture, comparing the results when stimulated by LPS and/or treated with PRS® CK STORM conditioned medium. For this purpose, the ELISA ATP Assay Kit Colorimetric (Ref: ab83355) from Abcam was used, and the Biorad iMark plate reader was used for reading.

#### **2.3 Generation of the** *in vivo* **inflammation model**

To perform the experimental model of acute lung injury, the experimental model described by Stephens et al. [51] was used. For this model, 8–10 weeks old male C57BL/6 mice were used and administered 5 mg/kg of bacterial lipopolysaccharide (LPS) in 50 μl of physiological solution retro-orbital under anesthesia. To decrease the possible suffering of the mice due to LPS, they were administered buprenorphine

hydrochloride in water at the established dose of 0.056 mg/ml. A total of 25 animals were used, with a number of animals per group of 5. The mice were conditioned 1 week prior to the procedure and were housed in standard conditions with access to food and water ad libitum with 12-h light/dark cycles at a temperature of 25°C and humidity greater than 40% over the course of the project.


The vehicle used in the vehicle control group is PBS (phosphate-buffered saline). The gold standard treatment consists of the administration of Anakinra (IL-1Ra) before and after inoculation with LPS. The test group received the established dose of imatinib 24 h prior to LPS administration. The animals were administered the drug every 24 h starting from the retro-orbital administration of LPS. Treatment was administered intravenously (40 μl), upon generation of the model and then every 24 h thereafter. Blood samples were taken 24, 48, and 72 h after generation of the model from the submaxillary sinus from which 120 μl were collected for hemodynamic and general biochemical study, which was performed with the Comprehensive Diagnostic Profile protocol (#500–0038), of the VetScan V2 device (Abaxis). After 72 h from the generation of the model, the animals were sacrificed and exsanguinated and plasma samples were collected for final biochemical and cytokine analysis. Specifically, TNF-α, IL-1β, and IL-6 as proinflammatory cytokines and IL-10 as anti-inflammatory cytokine were quantified. They were analyzed by Luminex: MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel – Immunology Multiplex Assay (Cat: MCYTOMAG-70 K) (Merck). During the study, hyperthermia/hypothermia, respiratory distress, weight loss, food and water consumption, as well as the existence of other behavioral disorders were monitored. After euthanasia and subsequent necropsy, the major organs (heart, lung, liver, kidney, and spleen) were removed and fixed and preserved in 4% formalin for histological study. A small portion of each organ, prior to fixation, was preserved directly by immersion in dry ice to study inflammatory cytokine content in future assays. All animals underwent Irwin's test every 24 h to obtain neuropharmacology data following the protocol of Mathiasen et al. [52].

#### **2.4 Statistical analysis**

The MTT and cytokine release assays, as well as the cytokine analysis of the culture supernatants, the biochemical values of the blood of the different groups of treated

*Immunomodulatory Effects of a M2-Conditioned Medium (PRS® CK STORM)… DOI: http://dx.doi.org/10.5772/intechopen.104486*

mice, and the values obtained in the qPCRs, were subjected to statistical analysis. For these, a two-tailed Student's t-test was performed to obtain the p-value between the different experimental groups and to analyze the existence of significant differences (p < 0.05). Statistical analysis was performed with Excel (Microsoft, Albuquerque, NM, USA).

#### **3. Results**

#### **3.1 Isolation of MSCs and monocytes**

The yield achieved in the isolation of MSCs was approximately 1 <sup>10</sup><sup>5</sup> cells per ml of lipoaspirate, and it was necessary to incubate for 16 days under the conditions described in the previous section to bring the culture to pass 4 (**Figure 1a**). The yield provided by the monocyte donor can be seen in **Table 3**.

Optical microscopy showed that the cell morphology of the adherent cells in the cultures corresponded to that of monocytes/macrophages.

The results of flow cytometric characterization of MSCs and monocytes from three co-cultures at the times studied are shown in **Figure 1**. Phenotypic characterization of MSCs shows the classic phenotype of CD90 > 90%, CD73 > 90%, CD31 < 2%, CD45 < 2%, and HLA-DR < 2%. The marker CD68 is used as macrophage identifier, CD163

**Figure 1.** *Percentage of positive cells for each antibody tested.*


**Table 3.**

*Data and yields obtained from monocyte donors.*

is mostly present on M2 type macrophages, and CD39 is expressed on macrophages/ monocytes in co-culture with MSC.

#### **3.2 Cytokine characterization of the conditioned medium**

The results of quantification by both ELISA and Multiplex are detailed in **Table 4**. To obtain the pattern (**Figure 2**), those molecules that could be quantified because they were within the detection limits of the method used in each case were studied, and statistically significant differences were sought with respect to the values taken as


*Immunomodulatory Effects of a M2-Conditioned Medium (PRS® CK STORM)… DOI: http://dx.doi.org/10.5772/intechopen.104486*


*Values are shown in picograms per milliliter. > and < indicate that the value is above or below the detection limits (respectively). SD: standard deviation; NP: not applicable.*

#### **Table 4.**

*Mean values of the molecules studied.*

#### **Figure 2.**

*Cytokine expression patterns. Shown are 16 mean values standard deviation of molecular characterization (MIP-1α, IL-6, IL-8, IL-10, IL-1Ra, RANTES, Leptine, HGF, MMP-3, MCP-1, Adiponectine, MMP-1, TRAIL, PDGF-BB, VEGF-A, IGF-1). (A) Secretome of monocytes; (B) secretome of co-culture. Stars mark values where there is a statistically significant difference (p < 0.05).*

control (conditioned medium of M2-like monocytes/macrophages). Those values that were significantly different in all the samples studied (five samples in triplicate) were considered to form a specific and reproducible pattern of monocyte secretome modification by co-culture with MSCs. To test whether the pattern obtained was specific to the cell type co-cultured with monocytes, the expression of the same secretome molecules was obtained under the same conditions, but co-culturing monocytes with the following cell types: osteocytes, chondrocytes, tenocytes, synoviocytes, myocytes, lymphatic vascular cells, and Schwann cells, was compared. From this comparison, eight different and characteristic secretomes could be specifically differentiated, quantifying a minimum of seven molecules: IL-6, leptin, HGF, MMP-1, MMP-3, adiponectin, and VEGF-A (data not shown).

#### **3.3 In vitro anti-inflammatory potency test**

THP-1 cells after 48 h of stimulation with PMA gain adherence to the culture plastic and take on a macrophage-like morphology. After 24 h of culture, the number

**Figure 3.**

*Graph a shows the results in terms of IL-1β release in the in vitro model of inflammation. Graph b shows the results in terms of TNF-α release in the in vitro model of inflammation. For these four studies, the results were analyzed in three independent experiments with two replicates of the analytical technique, for each type of experiment. Error bars indicate the standard deviation between samples. Asterisks (\*) mark values where there is a statistically significant difference (p < 0.05).*

of cells in suspension decreases and the number of cells adherent to the plastic increases, a symptom of correct differentiation. After 48 h, about 90% of the cells are adherent to the plastic and are used in the experimental model.

The in vitro inflammation model used in this research is based on stimulating the proinflammatory action of macrophages when exposed to LPS. **Figure 3** shows that the addition of our conditioned medium to the culture of THP-1 cells transformed into macrophages can reverse the effect of LPS on these macrophages, and a statistically significant difference can be observed, in addition to the difference observed with the use of soluble hydrocortisone. It can also be observed that the cells are sensitive to LPS stimuli at the concentration used.

#### **4. Biosafety and in vivo efficacy tests**

The results of the Irwin test are analyzed to evaluate at a general level the effect produced by the conditioned medium administered intravenously to mice, in which the cytokine storm model had been previously induced, by retrorbital injection of LPS. A slight decrease in temperature was observed in all LPS-injected groups. LPS administration was observed to induce changes in reflexes and behavior, such as hunching, piloerection, and tremors, which were increased throughout the 3-day trial. In contrast, exploratory activity, reaction to contact, and aggressiveness were slightly decreased. In all LPS-treated mice (with or without drug administration), mild diarrhea occurred, which in untreated or gold standard-treated mice resulted in more severe dehydration than in mice treated with the conditioned medium. In general, there is a trend that the symptoms caused by LPS administration are dissipated by treatment with our conditioned medium (PRS® CK STORM), demonstrating a beneficial effect on the mice without counterproductive effects (**Table 5**).

The variations observed have as reference value the parameters 1 day before generating the model. Mice 11, 18, and 22 died before completing the 3 days of the experiment.

From the blood obtained after euthanasia and exsanguination of the animals, biochemical tests were performed to determine the biochemical profiles, which are shown in **Table 6**. The Mann–Whitney test was used for statistical analysis, considering statistical significance as\*p < 0.05 in the case of statistically significant minor differences with respect to the baseline value and \*\*p < 0.05 in the case of statistically significant major differences with respect to the baseline value.

*Immunomodulatory Effects of a M2-Conditioned Medium (PRS® CK STORM)… DOI: http://dx.doi.org/10.5772/intechopen.104486*


#### **Table 5.**

*Results of the Irwin test; it includes the parameters studied on the third day after generating the model. Mice 11, 18 and 22 died before completing the 3 days of the experiment [52].*

**Figure 4** shows in percentage the relative variations observed at the posttreatment times (24, 48, and 72 h), with respect to the baseline values observed in each group.

The experimental model employed uses LPS as a causative agent of acute lung damage, causing a cytokine storm in the organism of mice like that produced by COVID-19 disease. We focused on the quantification of a small amount of these cytokines (TNF-α, IL-1β, IL-6, and IL-10) to evaluate the effect of the PRS® CK STORM. **Figure 5** shows the evolution of these cytokines detected in the sera of the mice on each of the days that the treatment lasted.

Histopathological analysis of the samples obtained from various organs of the mice obtained after the corresponding necropsies showed patchy lung thickening of the interstitium in a large part of the sample in the LPS treatment, while in the group treated with PRS® CK STORM it was observed that there was no lung damage, as in the control groups. Slight affectations were observed in liver and spleen, which the drug was also able to reverse. As for the heart and kidney, no pathological findings were detected. **Figure 6** shows examples of the lung sections studied in the different groups of the experiment.

#### **4.1 Mechanism of action study**

In order to approach the mechanism of action of our conditioned medium PRS® CK STORM, qPCR study is performed in order to analyze the effects of our complex biological drug under investigation on all common molecules involved in TLR2, TLR3, TLR4, TLR7, TLR8, TLR9, NOD1, NOD2, and NLRP3 pathways, TLR9, NOD1, NOD2, and NLRP3, and it is found that the conditioned medium downregulates the hyperactivity of these pathways, immunoregulating the key proteins involved in these pathways, being very remarkable the decrease of expression observed in TRAF6, caspase-1, RIPK1, IKKB, NF-kβ, MyD88, and NLRP3 proteins.

Following the method described in the corresponding section of this chapter, the total mRNA of the proteins named in the previous paragraph common to various pattern recognition pathways was extracted from THP-1m cells in culture used as control, from


#### *Purinergic System*


*Immunomodulatory Effects of a M2-Conditioned Medium (PRS® CK STORM)… DOI: http://dx.doi.org/10.5772/intechopen.104486*

> **Table 6.**

 *Results of the biochemical analysis of the mice, including the parameters studied pretreatment and posttreatment at 24, 48, and 72 h.*

#### **Figure 4.**

*Concentration values of the different metabolites that make up the biochemical profile of the mice, expressed as a percentage with respect to the baseline value observed in each group.*

#### **Figure 5.**

*Serum values of the different cytokines after 24, 48, and 72 h from the administration of the first treatment expressed in pg/ml as the mean of the values of the mice in each of the experimental groups. a: TNF-α. b: IL-1β. c: IL-6. d: IL-10. Values not shown are lower than the detection limit of the assay (2.8 pg/ml).*

those stimulated with LPS, and from those stimulated with LPS and treated with PRS® CK STORM, obtaining by qPCR the relative expression of the genes at the mRNA level normalized against GAPDH, the results of which are shown in **Figure 7**.

Similarly and under the same experimental methodology, the total mRNA of the pattern recognition proteins linked to main infectious processes TLR-2, TLR-3, TLR-4, and TLR-7 was extracted from THP-1m cells in culture used as control, from those stimulated with LPS and from those stimulated with LPS and treated with PRS® CK

*Immunomodulatory Effects of a M2-Conditioned Medium (PRS® CK STORM)… DOI: http://dx.doi.org/10.5772/intechopen.104486*

#### **Figure 6.**

*Pulmonary anatomopathological study in the different groups of mice in the experiment. It can be seen that the group treated with PRS® CK STORM (our conditioned medium), the appearance of the lung is very similar to that shown by the untreated control group. All sections have been stained with hematoxylin eosin. 1 images are taken in MO at 4 magnification and 2 images are taken in MO at 40 magnification.*

#### **Figure 7.**

*qPCR study showing the results of relative gene expression of proteins common to various pathways related to pattern recognition in infections, at the mRNA level normalized against GAPDH, considering statistical significance as \*p < 0.05.*

STORM, obtaining by qPCR the relative expression of the genes at mRNA level normalized against GAPDH, whose results are shown in **Figure 8**.

In order to estimate the possible action of our PRS® CK STORM conditioned medium through the purinergic system (**Figure 9**), the results of the ATP/ADP ratio

*qPCR study showing the results of relative gene expression of major pattern recognition receptor proteins in infections, at the mRNA level normalized against GAPDH, considering statistical significance as \*p < 0.05.*

#### **Figure 9.**

*(a) ATP/ADP ratio analysis in three groups of cell cultures (THP-1m,THP-1m + LPS, and THP-1m + LPS + PRS® CK STORM). Asterisks (\*) mark values where there is a statistically significant difference (p < 0.05). (b) Quantitative analysis of extracellular ATP in three groups of cell cultures (THP-1m,THP-1m + LPS, and THP-1m + LPS + PRS® CK STORM). Asterisks (\*) mark values where there is a statistically significant difference (p < 0.05). (c) qPCR study showing the results of the relative expression of purinergic A2a, A3, and P2X7 receptor genes at the mRNA level normalized against GAPDH, considering statistical significance as \*p < 0.05.*

obtained were analyzed comparatively among three groups with three replicates for each group, being the first group formed by THP-1m cells placed in culture alone, THP-1m cells stimulated with LPS at the doses described in Section 2, and the same THP-1m cells stimulated with LPS but in culture in the PRS® CK STORM conditioned medium (**Figure 9a**). The extracellular ATP contained in the same three groups of

*Immunomodulatory Effects of a M2-Conditioned Medium (PRS® CK STORM)… DOI: http://dx.doi.org/10.5772/intechopen.104486*

cultures with three replicates for each group was quantified (**Figure 9b**). Finally, following the method described in the corresponding section of this chapter, the total mRNA of purinergic receptors A2a, A3, and P2X7 was extracted from THP-1m cells in culture used as control, from those stimulated with LPS, and from those stimulated with LPS and treated with PRS® CK STORM, obtaining by qPCR the relative expression of the genes at the mRNA level normalized against GAPDH (**Figure 9c**).

#### **5. Discussion**

In the complex composition of this conditioned medium (PRS® CK STORM), all the growth factors, cytokines, and chemokines that are naturally produced by M2 type macrophages and MSCs, associated with innate immunity, are present, respecting the natural pleiotropic relationships, with an immunomodulatory cytokine profile from which a potent anti-inflammatory action is expected. In addition, according to the results obtained in test studies, the mechanism of action on TLR-7 receptors may possibly include some antiviral activity [32]. In fact, several experiments have shown that the secretomes of both cell types possess immunomodulatory properties. For example, direct injection of the supernatant of cultured mesenchymal stem cells (MSCs), which contains a variety of growth factors, prostaglandins and cytokines, can be applied to the treatment of kidney injury [45].

The theoretical advantage of using the complete conditioned medium versus some of its purified components lies in the synergistic mechanism between its different components [48], the result of subjecting the cell populations to a culture that, in vitro, attempts to emulate the immunomodulatory and regenerative microenvironment that occurs in vivo in diseased tissue. However, the main handicap of biological drugs with complex natural compositions will always be the variability between different batches and the practical impossibility of achieving a complete characterization, both qualitatively and quantitatively, as well as functionally [48]. Despite the earlier-mentioned, our group has been able to prove the existence of a stable cytokine pattern or fingerprint, which depends directly on the type of co-culture established and the conditions of the same and not on the donor of origin.

The anti-inflammatory capacity and potency shown by the PRS® CK STORM conditioned medium has been remarkable, showing statistically significant reductions in in vitro tests on TNF-α and IL-1β levels, these differences being very similar to those obtained with hydrocortisone. This anti-inflammatory immunomodulatory capacity, we have also been able to verify in the in vivo model, generated in mice. The mice treated with our PRS® CK STORM conditioned medium have shown normal behavior and the rest of the parameters analyzed in the Irwin test have been very similar to the control groups, where the cytokine storm had not been provoked. In fact, the comparative results in the in vivo test between the gold standard treatment used (Anakinra) and the PRS® CK STORM were clearly more favorable to the latter. It should be noted that in the group treated with PRS® CK STORM practically no pulmonary lesions were observed, while in the group treated with Anakinra inflammatory and fibrotic infiltrates were observed in a minimum of 30% of the surface of the sections. This coincides with the observation made at the time of sacrifice of the animals under study where both the control animals, in which the cytokine storm had not been provoked, and those treated with PRS® CK STORM, took more than 2 min to die in the CO2 chamber, while the animals treated with Anakinra died in 40 s and

those not treated in about 30 s, and these times can be directly related to the anatomopathological state observed at the pulmonary level.

The results obtained throughout this experiment suggest that the drug PRS® CK STORM is safe for intravenous administration, since no significant adverse effects have been observed in the different parameters analyzed, those found being mild. In addition, the drug significantly attenuates the detrimental effects caused by the cytokine storm associated with LPS administration. Most of the proteins and metabolites analyzed follow the same trend, regardless of the experimental group observed. With respect to albumin, the main protein present in blood, there is a decrease in albumin in all groups to which LPS was administered, which fits with that described by Ballmer et al. [53], with hypoalbuminemia occurring when the organism undergoes sepsis due to infection. Related to this, there is a decrease in total protein: a decrease in albumin will cause a decrease in total protein since the former is at very high concentrations. A decrease in blood glucose is also observed in all mice treated with LPS and LPS + gold standard. However, those treated with PRS® CK STORM managed to normalize the levels of glycemia, albuminemia, and total proteinemia 3 days after receiving the first dose. This fact was directly related to the clinical improvement observed in these animals subjected to the experimental treatment, given that they felt better than the rest of the mice, ate better, physical activity was normalized, and diarrhea was corrected. On the other hand, a significant increase in alanine amino transferase (ALT), total bilirubin, and amylase was observed during the experiment, which could be related to reactive hepatitis [54]. This increase was observed in all groups stimulated with LPS and was maintained in those treated with gold standard. However, in those treated with PRS® CK STORM, the figures normalized 72 h after the first treatment.

In all the groups where LPS was administered, there was a rapid increase in all the cytokines analyzed. From this, we can conclude that stimulation with bacterial lipopolysaccharide (LPS) is able to induce the expected inflammatory response, being more pronounced in more acute phase, the day after treatment administration and decreasing over time. In the control group and the vehicle, the presence of proinflammatory cytokines is not observed, which confirms that LPS is the cause of this response. However, in the group treated with LPS + gold standard, a lower increase of IL-1β is observed with respect to the rest of the groups where LPS was administered. This fact can be explained by the previous administration of the gold standard, recombinant IL-1Ra. However, despite the lower increase in this proinflammatory cytokine, the decrease in IL-1β finally achieved by the gold standard is even lower than that obtained with PRS® CK STORM treatment. In general terms, our PRS® CK STORM conditioned medium achieves greater control of all the cytokines analyzed in the experiment.

The results of the mechanism of action study show that PRS® CK STORM is able to immunomodulate in an anti-inflammatory way the expression of TLR-like pattern recognition pathways especially associated with infectious processes, such as TLR-2, TLR-3, TLR-4, and TLR-7. From the same study, it can be deduced that this effect is not only localized to these receptors but also acts at the level of various proteins common to these and other pathways, such as TRAF6, RIPK1, and IKKB, with the decrease in expression observed in the proteins NF-kβ, MyD88, caspase-1, and NLRP3 being very significant.

Many published studies have demonstrated the importance of the purinergic system in the inflammation associated with the cytokine storm caused by moderate/ severe infection, including COVID-19, and have shown that using various purinergic system receptors as a therapeutic target can limit the negative effects of the cytokine storm [21, 22, 55–57]. Extracellular ATP at high concentrations becomes a true alarmin [58], a potent proinflammatory signal capable of overexpressing and stimulating P2X-type purinergic receptors, especially P2X7R, located on various immune cells (neutrophils, eosinophils, monocytes, macrophages, mast cells, and lymphocytes) [59]. Extracellular adenosine triphosphate (eATP) is a well-characterized DAMP that modulates function and plasticity [60, 61]. This nucleotide can be released by stressed, injured, and dying cells or in response to TLR activation, reaching high concentrations in the extracellular medium [62].

In contrast, it has been shown that the balance between ATP and adenosine concentration is crucial in immune homeostasis. CD39 and CD73 are two ectonucleotidases that cooperate in the generation of extracellular adenosine by ATP hydrolysis, thus tipping the balance toward immunosuppressive microenvironments. Extracellular adenosine through A2A receptor stimulation has the ability to prevent activation and proliferation of both macrophages and T cells, thereby dramatically decreasing cytokine production [63].

From the results observed both in the cytometric characterization of M2 macrophages used in the co-culture to produce our conditioned medium, where a strong expression of CD39 and CD73 is observed, and from the study of the ATP/ADP ratio, where a clear increase of ADP is observed, we can deduce that one of the mechanisms of action of PRS® CK STORM is probably linked to the process of dephosphorylation of extracellular ATP, which is degraded by ectonucleotidases to adenosine, and the latter interacts with adenosine receptors, type A2A and A3, producing an immunomodulatory anti-inflammatory effect on the cytokine storm. This theory is supported by two further pieces of evidence; firstly by the statistically significant decrease observed in LPS-stimulated THP-1 cells treated with our PRS® CK STORM conditioned medium with respect to the levels observed in untreated LPS-stimulated THP-1 cells; and secondly by the combination of the observed reduction in the relative expression of P2X7 receptor mRNA with the observed increase in the relative expression of A3 and A2a receptor mRNA in LPS-stimulated THP-1 cells treated with PRS® CK STORM relative to untreated THP-1 cells.

In all assays used in the in vitro and in vivo models, both employed LPS as an inducer of inflammation. Although the immunomodulatory mechanisms induced by bacterial antigens with respect to viral antigens in immune cells are different at the mechanistic level, the inflammatory response at the level of innate immunity ends up sharing numerous points in common [64, 65]. Therefore, the effect that PRS® CK STORM has on these models of inflammation with LPS can be exportable to what can happen, at the level of immune response, during a viral infection. In fact, it has been shown that monocytes and macrophages stimulated with LPS and ATP increase the release of IL-1β [66, 67].

All these results observed in the study on the possible mechanism of action of our complete conditioned medium (PRS® CK STORM) demonstrate that the immunomodulatory anti-inflammatory effect observed is a direct consequence of the action of various molecules contained in this medium, acting in a synergistic and pleiotropic manner on various therapeutic targets associated with different proinflammatory pathways, managing to downregulate the activation of the inflammasome, the inflammatory activation of the purinergic system, the activation of various pathways of pattern recognition associated with infections, etc., thus avoiding the possible feedback effects that therapeutic approaches based on the inhibition of a single receptor or a single inflammatory pathway may have.

#### **6. Conclusions**

The co-culture of M2 macrophages with MSCs allows the simple production of a complete conditioned medium (PRS® CK STORM), which has a clear antiinflammatory profile. In line with this characterization, it has been demonstrated that PRS® CK STORM is able to stop macrophage overactivation in an in vitro inflammation model, through several mechanisms, including the expression pattern of TLR's and the purinergic system. Likewise, the action capacity of this drug has also been demonstrated in vivo, by improving the symptomatology and tissue damage induced in mice in another model of inflammation. Therefore, PRS® CK STORM is proposed as an effective and safe treatment to treat cytokine storms associated with moderate/ severe infectious processes of any etiology, including that associated with COVID-19.

### **Author details**

Juan Pedro Lapuente SCO of the Living Cells Molecular and Cellular Biology Research Laboratory of the Fuenlabrada Universitary Hospital, Madrid, Spain

\*Address all correspondence to: jplapuente@yahoo.es

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Immunomodulatory Effects of a M2-Conditioned Medium (PRS® CK STORM)… DOI: http://dx.doi.org/10.5772/intechopen.104486*

#### **References**

[1] Weiss U. Nature insight: Inflammation. Nature. 2002;**420**:845

[2] Medzhitov R. Origin and physiological roles of inflammation. Nature. 2008;**454**:428-435

[3] Risdall RJ, McKenna RW, Nesbit ME, Krivit W, Balfour HH Jr, Simmons RL, et al. Virus-associated hemophagocytic syndrome: A benign histiocytic proliferation distinct from malignant histiocytosis. Cancer. 1979;**44**:993-1002

[4] Guo XJ, Thomas PG. New fronts emerge in the influenza cytokine storm. Seminars in Immunopathology. 2017;**39** (5):541-550. DOI: 10.1007/s00281-017- 0636-y

[5] Tseng Y-T, Sheng W-H, Lin B-H, Lin C-W, Wang J-T, Chen Y-C, et al. Causes, clinical symptoms, and outcomes of infectious diseases associated with hemophagocytic lymphohistiocytosis in Taiwanese adults. Journal of Microbiology, Immunology, and Infection. 2011;**44**(3):191-197

[6] Cascio A, Pernice LM, Barberi G, Delfino D, Biondo C, Beninati C, et al. Secondary hemophagocytic lymphohistiocytosis in zoonoses. A systematic review. European Review for Medical and Pharmacological Sciences. 2012;**16**:1324-1337

[7] Singh ZN, Rakheja A, Yadav TP, Shome J. Infection-associated haemophagocytosis: The tropical spectrum. Clinical and Laboratory Haematology. 2005;**27**(5):312-315

[8] Teijaro JR. Cytokine storms in infection diseases. Seminars in Immunopathology. 2017;**39**:501-503

[9] Himanshu K, Taro K, Shizuo A. Pathogen recognition by the innate immune system. International Reviews of Immunology. 2011;**30**(1):16-34

[10] Taro K, Shizuo A. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity. 2011;**34**(5):637-650

[11] Komal D, Manoj B, Gourango B, Atul B, Sangita B. TLRs/NLRs: Shaping the landscape of host immunity. International Reviews of Immunology. 2011;**37**(1):3-19

[12] Martinon F, Burns K, Tschopp J. The inflammasome: A molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Molecular Cell. 2002;**10**(2):417-426

[13] Root-Bernstein R. Synergistic activation of toll-like and NOD receptors by complementary antigens as facilitators of autoimmune disease: Review, model and novel predictions. International Journal of Molecular Sciences. 2020;**21**:4645

[14] Hosseini AM, Majidi J, Baradaran B, Yousefi M. Toll-like receptors in the pathogenesis of autoimmune diseases. Advanced Pharmaceutical Bulletin. 2015; **5**:605-614

[15] Dabbagh K, Lewis DB. Toll-like receptors and T-helper-1/T-helper-2 responses. Current Opinion in Infectious Diseases. 2003;**16**:199-204

[16] Tukhvatulin AI, Gitlin II, Shcheblyakov DV, Artemicheva NM, Burdelya LG, Shmarov MM, et al. Combined stimulation of toll-like receptor 5 and NOD1 strongly potentiates activity of NF-kβ, resulting in enhanced innate immune reactions and resistance to *Salmonella enterica*

Serovar Typhimurium infection. Infection and Immunity. 2013;**81**: 3855-3864

[17] Moreira LO, Zamboni DS. NOD1 and NOD2 signaling in infection and inflammation. Frontiers in Immunology. 2012;**3**:328

[18] Olbei M, Hautefort I, Modos D, Treveil A, Poletti M, Gul L, et al. SARS-CoV-2 causes a different cytokine response compared to other cytokine storm-causing respiratory viruses in severely ill patients. Frontiers in Immunology. 2021;**1**(12):629193

[19] Root-Bernstein R. Innate receptor activation patterns involving TLR and NLR synergisms in COVID-19, ALI/ ARDS and sepsis cytokine storms: A review and model making novel predictions and therapeutic suggestions. International Journal of Molecular Sciences. 2021;**22**:2108

[20] Simões JLB, Bagatini MD. Purinergic signaling of ATP in COVID-19 associated Guillain–Barré syndrome. Journal of Neuroimmune Pharmacology. 2021; **16**(1):48-58

[21] Franciosi MLM, Lima MDM, Schetinger MRC, Cardoso AM. Possible role of purinergic signaling in COVID-19. Molecular and Cellular Biochemistry. 2021;**476**(8):2891-2898

[22] Simões JLB, de Araújo JB, Bagatini MD. Anti-inflammatory therapy by cholinergic and purinergic modulation in multiple sclerosis associated with SARS-CoV-2 infection. Molecular Neurobiology. 2021;**58**(10): 5090-5111

[23] Abbracchio MP, Burnstock G. Purinoceptors: Are there families of P2X and P2Y purinoceptors? Pharmacology & Therapeutics. 1994;**64**:445-475

[24] Di Virgilio F, Dal Ben D, Sarti AC, Giuliani AL, Falzoni S. The P2X7 receptor in infection and inflammation. Immunity. 2017;**47**:15-31

[25] Di Virgilio F, Schmalzing G, Markwardt F. The elusive P2X7 macropore. Trends in Cell Biology. 2018; **28**:392-404

[26] Illes P, Rubini P, Ulrich H, Zhao Y, Tang Y. Regulation of microglial functions by purinergic mechanisms in the healthy and diseased CNS. Cell. 2020;**9**:1108

[27] Csoka B, Németh ZH, Törö G, Idzko M, Zech A, Koscsó B, et al. Extracellular ATP protects against sepsis through macrophage P2X7 purinergic receptors by enhancing intracellular bacterial killing. The FASEB Journal. 2015;**29**: 3626-3637

[28] Wu X, Ren J, Chen G, Wu L, Song X, Li G, et al. Systemic blockade of P2X7 receptor protects against sepsis-induced intestinal barrier disruption. Scientific Reports. 2017;**7**:4364

[29] Di Virgilio F, Tang Y, Sarti AC, Rossato M. A rationale for targeting the P2X7 receptor in coronavirus disease 19. British Journal of Pharmacology. 2020; **177**(21):4990-4994

[30] Ren W, Rubini P, Tang Y, Engel T, Illes P. Inherent P2X7 receptors regulate macrophage functions during inflammatory diseases. International Journal of Molecular Sciences. 2021;**23** (1):232

[31] Carty F, Mahon BP, English K. The influence of macrophages on mesenchymal stromal cell therapy: Passive or aggressive agents? Clinical and Experimental Immunology. 2017;**188**(1): 1-11

*Immunomodulatory Effects of a M2-Conditioned Medium (PRS® CK STORM)… DOI: http://dx.doi.org/10.5772/intechopen.104486*

[32] Asami T, Ishii M, Fujii H, Namkoong H, Tasaka S, Matsushita K, et al. Modulation of murine macrophage TLR7/8-mediated cytokine expression by mesenchymal stem cell-conditioned medium. Mediators of Inflammation. 2013;**2013**:264260

[33] Vallés G, Bensiamar F, Crespo L, Arruebo M, Vilaboa N, Saldaña L. Topographical cues regulate the crosstalk between MSCs and macrophages. Biomaterials. 2015; **37**:124-133

[34] English K. Mechanisms of mesenchymal stromal cell immunomodulation. Immunology and Cell Biology. 2013;**91**(1):19-26

[35] Deng Y, Zhang Y, Ye L, Zhang T, Cheng J, Chen G, et al. Umbilical cord-derived mesenchymal stem cells instruct monocytes towards an IL10 producing phenotype by secreting IL6 and HGF. Scientific Reports. 2016;**6**: 37566

[36] Melief SM, Geutskens SB, Fibbe WE, Roelofs H. Multipotent stromal cells skew monocytes towards an antiinflammatory interleukin-10-producing phenotype by production of interleukin-6. Haematologica. 2013;**98**:888-895

[37] Saldaña L, Bensiamar F, Vallés G, Mancebo FJ, García-Rey E, Vilaboa N. Immunoregulatory potential of mesenchymal stem cells following activation by macrophage-derived soluble factors. Stem Cell Research & Therapy. 2019;**10**(1):58

[38] Luz-Crawford P, Djouad F, Toupet K, Bony C, Franquesa M, Hoogduijn MJ, et al. Mesenchymal stem cell-derived interleukin 1 receptor antagonist promotes macrophage polarization and inhibits B cell differentiation. Stem Cells. 2016;**34**:483-492

[39] Ge W, Jiang J, Arp J, Liu W, Garcia B, Wang H. Regulatory T-cell generation and kidney allograft tolerance induced by mesenchymal stem cells associated with indoleamine 2,3-dioxygenase expression. Transplantation. 2010;**90**: 1312-1320

[40] Jiang XX, Zhang Y, Liu B, Zhang SX, Wu Y, Yu XD, et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood. 2005;**105**:4120-4126

[41] Ramasamy R, Fazekasova H, Lam EW, Soeiro I, Lombardi G, Dazzi F. Mesenchymal stem cells inhibit dendritic cell differentiation and function by preventing entry into the cell cycle. Transplantation. 2007;**83**:71-76

[42] Jackson MV, Morrison TJ, Doherty DF, McAuley DF, Matthay MA, Kissenpfennig A, et al. Mitochondrial transfer via tunneling nanotubes is an important mechanism by which mesenchymal stem cells enhance macrophage phagocytosis in the in vitro and in vivo models of ARDS. Stem Cells. 2016;**34**:2210-2223

[43] Gonçalves FDC, Luk F, Korevaar SS, Bouzid R, Paz AH, López-Iglesias C, et al. Membrane particles generated from mesenchymal stromal cells modulate immune responses by selective targeting of pro-inflammatory monocytes. Scientific Reports. 2017;**7**:12100

[44] Burnstock G, Boeynaems JM. Purinergic signalling and immune cells. Purinergic Signal. 2014;**10**:529-564

[45] Iseri K, Iyoda M, Ohtaki H, Matsumoto K, Wada Y, Suzuki T, et al. Therapeutic effects and mechanism of conditioned media from human mesenchymal stem cells on anti-GBM glomerulonephritis in WKY rats.

American Journal of Physiology. Renal Physiology. 2016;**310**(11):F1182-F1191

[46] Shen Y, Song J, Wang Y, Chen Z, Zhang L, Yu J, et al. M2 macrophages promote pulmonary endothelial cells regeneration in sepsis-induced acute lung injury. Annals of Translational Medicine. 2019;**7**(7):142

[47] Dahbour S, Jamali F, Alhattab D, Al-Radaideh A, Ababneh O, Al-Ryalat N, et al. Mesenchymal stem cells and conditioned media in the treatment of multiple sclerosis patients: Clinical, ophthalmological and radiological assessments of safety and efficacy. CNS Neuroscience & Therapeutics. 2017;**23** (11):866-874

[48] Laggner M, Gugerell A, Bachmann C, Hofbauer H, Vorstandlechner V, Seibold M, et al. Reproducibility of GMP-compliant production of therapeutic stressed peripheral blood mononuclear cell-derived secretomes, a novel class of biological medicinal products. Stem Cell Research & Therapy. 2020;**11**(1):9

[49] Lapuente JP, Dos-Anjos S, Blázquez-Martínez A. Intra-articular infiltration of adipose-derived stromal vascular fraction cells slows the clinical progression of moderate-severe knee osteoarthritis: Hypothesis on the regulatory role of intra-articular adipose tissue. Journal of Orthopaedic Surgery and Research. 2020;**15**(1):137

[50] Park EK, Jung HS, Yang HI, Yoo MC, Kim C, Kim KS. Optimized THP-1 differentiation is required for the detection of responses to weak stimuli. Inflammation Research. 2007;**56**(1): 45-50

[51] Stephens RS, Johnston L, Servinsky L, Kim BS, Damarla M. The tyrosine kinase inhibitor imatinib prevents lung

injury and death after intravenous LPS in mice. Physiological Reports. 2015;**3**(11): e12589

[52] Mathiasen JR, Moser VC. The Irwin test and functional observational battery (FOB) for assessing the effects of compounds on behavior, physiology, and safety pharmacology in rodents. Current Protocols in Pharmacology. 2018;**83**(1):e43

[53] Ballmer PE, Ochsenbein AF, Schütz-Hofmann S. Transcapillary escape rate of albumin positively correlates with plasma albumin concentration in acute but not in chronic inflammatory disease. Metabolism. 1994;**43**(6): 697-705

[54] Neurath MF. COVID-19: Biologic and immunosuppressive therapy in gastroenterology and hepatology. Nature Reviews. Gastroenterology & Hepatology. 2021;**18**(10):705-715

[55] Leão Batista Simões J, Fornari Basso H, Cristine Kosvoski G, Gavioli J, Marafon F, Elias Assmann C, et al. Targeting purinergic receptors to suppress the cytokine storm induced by SARS-CoV-2 infection in pulmonary tissue. International Immunopharmacology. 2021;**100** (108150)

[56] Dos Anjos F, Simões JLB, Assmann CE, Carvalho FB, Bagatini MD. Potential therapeutic role of purinergic receptors in cardiovascular disease mediated by SARS-CoV-2. Journal of Immunology Research. 2020;**1** 8632048

[57] Hasan D, Shono A, van Kalken CK, van der Spek PJ, Krenning EP, Kotani T. A novel definition and treatment of hyperinflammation in COVID-19 based on purinergic signalling. Purinergic Signal. 2022;**18**(1):13-59

*Immunomodulatory Effects of a M2-Conditioned Medium (PRS® CK STORM)… DOI: http://dx.doi.org/10.5772/intechopen.104486*

[58] Lai CC, Shih TP, Ko WC, Tang HJ, Hsueh PR. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): The epidemic and the challenges. International Journal of Antimicrobial Agents. 2020;**55**(3):105924

[59] Liu P, Chen W, Chen JP. Viral metagenomics revealed Sendai virus and coronavirus infection of Malayan pangolins (*Manis javanica*). Viruses. 2019;**11**(11):979

[60] Barberà-Cremades M, Baroja-Mazo A, Pelegrín P. Purinergic signaling during macrophage differentiation results in M2 alternative activated macrophages. Journal of Leukocyte Biology. 2016;**99**(2):289-299

[61] Savio LEB, Coutinho-Silva R. Immunomodulatory effects of P2X7 receptor in intracellular parasite infections. Current Opinion in Pharmacology. 2019;**47**:53-58

[62] Cohen HB, Briggs KT, Marino JP, Ravid K, Robson SC, Mosser DM. TLR stimulation initiates a CD39-based autoregulatory mechanism that limits macrophage inflammatory responses. Blood. 2013;**122**(11):1935-1945

[63] Bono MR, Fernández D, Flores-Santibáñez F, et al. CD73 and CD39 ectonucleotidases in T cell differentiation: Beyond immunosuppression. FEBS Letters. 2015; **589**:3454-3460

[64] Finlay BB, McFadden G. Antiimmunology: Evasion of the host immune system by bacterial and viral pathogens. Cell. 2006;**124**(4):767-782

[65] Goldstein ME, Scull MA. Modeling innate antiviral immunity in physiological context. Journal of Molecular Biology. 2021;**1**:167374

[66] Ferrari D, Chiozzi P, Falzoni S, Dal Susino M, Melchiorri L, Baricordi OR, et al. Extracellular ATP triggers IL-1 beta release by activating the purinergic P2Z receptor of human macrophages. Journal of Immunology. 1997;**159**(3):1451-1458

[67] Perregaux DG, McNiff P, Laliberte R, Conklyn M, Gabel CA. ATP acts as an agonist to promote stimulus-induced secretion of IL-1 beta and IL-18 in human blood. Journal of Immunology. 2000;**165**(8):4615-4623

#### **Chapter 8**

## The Potential of the Purinergic System as a Therapeutic Target of Natural Compounds in Cutaneous Melanoma

*Gilnei Bruno da Silva, Daiane Manica, Marcelo Moreno and Margarete Dulce Bagatini*

#### **Abstract**

Cutaneous melanoma is an aggressive and difficult-to-treat disease that has rapidly grown worldwide. The pharmacotherapy available in so many cases results in low response and undesirable side effects, which impair the life quality of those affected. Several studies have been shown that the purinergic system is involved in cancer context, such as in cutaneous melanoma. With technological advances, several bioactive compounds from nature are studied and presented as promising adjuvant therapies against cancer, as phenolic compounds and related action by purinergic system modulations. Thus, phenolic compounds such as rosmarinic acid, resveratrol, tannic acid, as well as vitamin D may be promising substances in a therapeutic perspective to treat cutaneous melanoma *via* purinergic system pathway. More research needs to be done to open up new horizons in the treatment of melanoma by the purinergic signaling.

**Keywords:** purinergic signaling, skin cancer, adjuvant therapies, therapeutic target

#### **1. Introduction**

Cutaneous melanoma (CM) is a disease that arises in transition of dermis and epidermis, where the melanocytes are localized. The melanogenesis process starts due to DNA damage secondary to a UV exposition, which can be chronic or acute intermittent exposure. Furthermore, other risk factors are associated with melanoma as frequency of sunbathing, ultraviolet A exposure, low skin phototypes, atypical nevus syndrome, skin sunburn events mostly during childhood and adolescence, a large number of skin moles (congenital or not), familiar or personal history of CM or skin cancer not melanoma. The DNA damage alters the proliferation and cell cycle, culmining in dysregulated apoptosis mechanisms. The CM is characterized by the high invasiveness, a high metastatic capacity, causing a short survival period and high mortality rates due to pharmacological resistance [1, 2].

For the systemic treatment of the patient with high-risk disease to metastasis, pharmacotherapy is used with drugs that can manifest collateral effects, in addition to presenting inefficient mechanisms to guarantee the survival of patients [3]. In this sense, several literatures have indicated biochemical therapy as a promising adjuvant in the CM management [4], even so it is urgent researchers to develop new options for a rise in patients' survival [1]. Therefore, many research science teams have been engaged to discover melanoma treatment and an interesting alternative to this would be to use natural substances, such as phenolic compounds that can have anticancer effects [5].

An efficient and rapid diagnosis is a priority among the medical and scientific community [6]. Furthermore, correctly and effectively pharmacological therapy promotes better prognosis and better quality of life for the melanoma cutaneous illness. Taking into consideration, the aim of this chapter was to provide an overview of potential modulations of the purinergic system in the treatment of cutaneous melanoma. Thus, initially, the cutaneous melanoma will be characterized based on epidemiology and therapeutics, as well as the purinergic system with its details, and finally, show that some natural compounds have potential in modulating purinergic signaling in melanoma.

#### **2. Cutaneous melanoma: Epidemiology and current therapeutic treatments**

Cutaneous melanoma is a disease that has a wide spectrum in relation to the prognosis of the patient who develops this disease. The diagnosis can be made from when the neoplasm is still restricted to the epidermis (*in situ*) in the disseminated form, where the malignant disease can affect other organs [7]. Data from the latest Global Cancer Statistics survey estimated for the year 2020 that cutaneous melanoma represents 1.7% of all malignant neoplasms, corresponding to 324,635 new cases worldwide. The number of deaths due to melanoma was 57,043. Comparing these numbers with the previous survey (2018), there was a considerable increase in the number of new cases, which was 232,100, as well as the number of deaths recorded (55,550 deaths). Both incidence and mortality rates differ according to each region of the planet, in addition to regions within the same country. Oceania has the highest ratio of number of cases; in Australia for the year 2020, it was expected 1 case of melanoma for every 104 male inhabitants and 1 case for every 185 females [8].

Also, North America and some European countries have high incidence rates of this disease. In the United States alone, 99,780 new cases and 7650 deaths from melanoma are expected in 2022 [9]. In the Scandinavian region, the incidence ranges from 15 to 18 cases/100,000 inhabitants; Northern European countries vary from 12 to 28 cases/100,000 inhabitants [10]. In Asia, Africa, and South America, the number of cases is lower, although there are regional differences as is the case in Brazil, where the South and Southeast regions have the highest number of cases when compared with other areas of the country [11–14].

Early detection of cutaneous melanoma is a fundamental factor in reducing mortality. Individuals diagnosed in the early stages have a 98% survival rate, while those diagnosed in advanced stages have a significantly decreased survival rate—between 63.8 and 15%. Patients diagnosed with stage III and IV melanoma have survival rates (5 years) of 70% and 30%, respectively. For most human malignancies, the use of chemotherapy for systemic treatment changed the natural history of the disease, and in melanoma the reality, until recently, was different with response rates comparable to the use of placebo [15]. Since the introduction of chemotherapy for adjuvant

*The Potential of the Purinergic System as a Therapeutic Target of Natural Compounds… DOI: http://dx.doi.org/10.5772/intechopen.105457*

treatment of malignant neoplasms, numerous therapeutic regimens have been tried in patients with metastatic melanoma [16, 17].

The most widely used treatment for patients with disseminated disease was dacarbazine, but only about 15–20% had some degree of response and 2% were still alive after 5 years of follow-up: a response rate comparable to the placebo-treated group of patients in the early clinical trials [15]. The use of high-dose interleukin-2 (IL-2) was the first treatment that changed the natural history of a small portion of patients with stage IV melanoma, but resulted in severe side effects that often affected survival [18, 19]. In the following years, molecules with direct action on pathways were responsible for controlling cell growth and division emerged, which were called "targeted therapy," such as BRAF and MEK inhibitors [19, 20]. However, only part of the melanoma patients can benefit from these inhibitors, because it is necessary that the genes involved in these pathways are mutated in order to get a response [21].

With the development of immunotherapy, other molecules called "immune system checkpoint inhibitors", such as pembrolizumab and nivolumab (programmed cell death protein-1 [PD-1] inhibitors), and ipilimumab (cytotoxic T lymphocyte antigen-4 [CTLA-4] inhibitor), have been routinely used as adjuvant and neoadjuvant treatments in melanoma patients with prognostic factors associated with poor survival [22, 23]. However, only 20% of patients show complete and lasting response with this type of therapy, and no biomarkers have been defined yet that can predict who will benefit from the use of these drugs [23–25].

#### **3. Evidences that purinergic system plays a role in cutaneous melanoma**

The purinergic system is a sophisticated cell-cell communication and ubiquitously expressed in the human body that orchestrates numerous cellular responses in the context of health and disease, displaying several biological processes. Most discussed extracellular signaling molecules include nucleotides such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and the nucleoside adenosine (Ado) [26–30]. The components belonging to this system are divided between receptors, according to the signaling molecule, as well as in enzymes. Thus, the P2 receptor is sensitized by adenine nucleotides, such as ATP, ADP, and AMP, being subdivided into P2X (1–7) and P2Y (1–12), while the P1 group is signaled Ado molecules and differentiated into A1, A2, A2B, A3 [31]. The levels of signaling molecules are controlled by enzymes known as ectonucleotidases, expressed on the surface of cells. They are nucleoside triphosphate diphosphohydrolase 1 (NTPDase-1/ CD39), 5′-nucleotidase (5'-NT/CD73), and adenosine deaminase (ADA) enzymes, which metabolize ATP/ADP into AMP, AMP into adenosine (Ado), and finally this into inosine, respectively [32–35] (**Figure 1**).

In this context, it is well known that this system is involved in cancer dynamics and has a close relationship with the immune responses, such as in lung cancer [36, 37], leukemia [38], cutaneous melanoma [30, 39], pancreatic cancer [40], and gastric cancer [41]. Likewise, it has been reported that ATP is considered a pivotal molecule, which largely influences immune responses in peripheral and central tissues, can be released from the inflammatory and tumor cells *via* different mechanisms, such as exocytosis, plasma-membrane channels pannexin, or lysis, and getting accumulated in tumor microenvironment (TME) [42–46].

Thus, yet few studies have been performed to understand the involvement of purinergic signaling in melanoma, some robust evidence showed that this cell pathway

#### **Figure 1.**

*Purinergic system components and functions. Adenosine triphosphate (ATP), key molecule of the purinergic system, can be released in an extra-cell environment and act as agonist on P2XR and P2YR. Furthermore, the ectonucleotidases presenting on cell surface are capable to hydrolyze the ATP into others nucleotides, such as adenosine monophosphate (AMP), adenosine diphosphate (ADP), and nucleosides, such as adenosine (ado). Between the purinergic receptors, only P1 (P1R) have Ado as agonist. The ectonucleotidase pyrophosphatase/ phosphodiesterase (E-NPPS) has potential to break ATP straight to AMP, whereas ectonucleoside triphosphate diphosphohydrolase (E-NTPDase-CD39) can break ATP to ADP or ADP to AMP. In this enzyme orchestra, only one ectoenzyme is capable of hydrolyzing AMP to Ado, the ecto-5*′*-nucleotidase (E-5'-NT-CD73). In the end of the purinergic cascade, the adenosine deaminase (ADA) converts Ado to inosine (Ino). Source: The authors (2022).*

plays an important role in this disease. In this sense, a component-system widely reported is the P2X7 receptor, which is expressed by cancer cells, as in melanoma, and is mostly associated with tumor cell killing *via* ATP key molecule signaling modulation [47–49]. However, if hydrolyzed to ADP can present an immunosuppression effect [39], as well as Ado, a nucleoside product of ATP hydrolysis that mediates the protective response, such as immunosuppressive and anti-inflammatory effects on healthy tissues, has a pro-carcinogenic property on melanoma [50–53]. Data about the role of ATP in melanoma patients were found by Mânica et al. [50], who indicated that an increased inflammatory process by extracellular ATP leads to an immunosuppressive profile even after surgical removal, which, in turn, corroborated previous information.

Given the pleiotropic actions, the role of P2X7R depends on the nucleotide receptor-interactions, as well as concentration, being that these interactions can promote or inhibit melanoma. Taking account, recently it was reinforced that P2X7 is overexpressed in patients affected by metastatic malignant melanoma and that its expression closely correlates with reduced overall survival. This is because P2X7 stimulation is capable of miRNA-containing microvesicles and exosomes from melanoma cells [54]. Furthermore, it was hypothesized that the Warburg effect is possibly linked to P2X7 modulation by ATP in melanoma. Once activated by ATP, the PI3K-AKT pathway upregulates glycolytic cascade enzymes, which promotes lactate generation and acidification of the TME. Acidification of extracellular microenvironment alters immune response and supporting cancer [5].

Conversely, Hattori et al. [55] by treatment of B16 melanoma cells with oxidized ATP (oxATP) found significantly decreased cell proliferation at concentrations between 300 and 500 mM in low pH conditions. From this, they proposed that the P2X7R is a promising target for treatment of solid tumors. The same way, White

#### *The Potential of the Purinergic System as a Therapeutic Target of Natural Compounds… DOI: http://dx.doi.org/10.5772/intechopen.105457*

et al. [56] after an experimental incubation of melanoma cells with P2X7-agonist 2′–3′-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate found decrease in cell number. In the immunological scenario, P2X7 activity has been associated with tumorinfiltrating T cells (TILs), which leads to senescence and limits tumor suppression, in addition to affected cell cycling of effector T cells and resulting in generation of mitochondrial reactive oxygen species (ROS) and p38 MAPK-dependent upregulation of cyclin-dependent kinase inhibitor 1A [57].

Although the P2X are widely related, other receptors also have been involved in melanoma disease. The P2Y1 receptor was indicated as potential to reduce melanoma cell proliferation; however, P2Y2 usually appears to increase cell numbers [58]. Still, the P2Y12 seems to promote tumor metastasis by platelet activation in melanoma cells [59, 60]. Interestingly, one factor, which leads to skin cancer, UV-B irradiation, seems to have a relationship with purinergic signaling, and severe effects have been associated between irradiation type and reduced P2X1 and P2Y2 receptors, as well as to destruction of P2X7 receptors, with the possibility of contributing to malignant transformation of keratinocytes [61].

From the Ado-stimulated receptor perspective, the A2AR and A3AR seem to lead to melanoma cells' death *via* proliferation mechanisms. Deletion of A2ARs was capable of reverting immunosuppression in B16-melanoma-bearing mice, immune cells responses [62–64]. Koszałka et al. [65] also showed that A1R, A2AR, and A3R receptors play an important role in melanoma (B16 type cells) by modulating angiosupport and immunosuppression in mice. An interesting study performed with melanoma cells discovered that A2B receptor blockade can impair IL-8 production, whereas blocking A3 receptors, it is possible to further decrease VEGF secretion in melanoma cells treated with etoposide (VP-16) and doxorubicin. Thus, treatment of melanoma cells with the DNA-damaging drugs such as VP-16 and doxorubicin resulted in Ado receptors modulations and chemotherapeutic potential [66].

On the other side, ectonucleotidases that control the purinergic chain also are involved in neoplasias. The CD39 decreased activity mitigates ATP hydrolysis, leading to extracellular accumulation of this nucleotide. This was evidenced by Manica et al. [50] that showed that post-surgery CM patients present high ATP levels in microenvironment compared with the healthy controls, suggesting being the cause of poor prognosis [50]. Thus, the ectonucleotidases action seems to play an important role in cancer context as well as in other purinergic components.

Although the CD39 and CD73 dynamics are responsible for forming most Ado extracellular content, another enzyme involved is the E-NPP, which hydrolyzes AMP to Ado. Several studies have evidenced that these ectoenzymes are increased in cancer context [45, 67–69]. In melanoma, studies suggested high expression of the CD73 in patients [70–72]. Also, in the melanoma mouse model, a CD73 inhibitor improved T and B cell-mediated antitumor immunity and reduced tumor growth [73]. The hydrolysis CD73 capacity is known and involved in melanoma; however, recently a nonenzymatic action of this enzyme was related, playing a role in cell migration on extracellular matrix through focal adhesion kinase (FAK) [74].

#### **4. Natural compounds with antitumor effect and their purinergic system relationship**

Considering the need for new therapies and therapeutic targets for the treatment of cutaneous melanoma, studies with compounds that modulate the purinergic

system and have antitumor effects have been carried out, as is the case with phenolic compounds and vitamin D [75, 76]. **Figure 2** represents some possible modulatory mechanisms of natural compounds on the purinergic system in the CM context.

Phenolic compounds are secondary metabolites present in plants, whose function is to participate in their development and protect them from pathogens and UV radiation [77]. More than 8000 compounds have been identified so far, and most of these compounds have some beneficial property to humans [78]. Their therapeutic actions are related to the structure, mainly to the phenolic rings, in which these compounds are classified by the number of rings and structural elements they have, forming four major groups: phenolic acids, stilbenes, lignans, and flavonoids [79]. The most cited group of polyphenols in the literature with therapeutic actions including antitumor effect is flavonoids, which are present in foods consumed daily such as fruits, vegetables, vegetables, red wine, coffee, and green tea [80–82].

Resveratrol (3,5,4-trihydroxy-trans-stilbene) is found in red wine and in the skin of dark grapes, a polyphenol with antitumor activity, considered a candidate for the treatment of cutaneous melanoma, which has been shown to modulate the expression and activity of CD73, ADA enzyme, and P2X7 and A2A receptors, which are closely related to tumor progression [83–86]. Tannic acid, a polyphenol, was shown to be able to induce cell death in several types of cancer cells, such as cutaneous melanoma, prostate cancer, glioblastoma [87, 88].

Thus, Bona et al. [89] tested the antitumor effect of this substance in rats with glioblastoma and the interference with ectonucleotidases, in which tannic acid was able to increase the hydrolysis of ATP and AMP nucleotides and decrease the

#### **Figure 2.**

*Purinergic system modulation in cutaneous melanoma by means of natural compounds. The literature has been evidenced in the adjuvant therapeutic perspective that several compounds derived from nature can act against carcinogenesis and cutaneous melanoma. Resveratrol, from the grape, has the potential both to increase CD73 expression and modulate P2X7 receptors, which control the growth progression and metastasis. Rosmarinic acid, a phenolic acid from rosemary, can block the P2X7 receptor and inhibits the agonist mechanism by ATP, as well as have antagonism ADP-like on the P2Y12 receptor, leading to a decrease of tumor mass formation. Two important derivatives of coffee, the caffeic acid and caffeine, have been shown as interesting modulators of P1 receptors. These adenosinergic receptors are intimately related to tumor immunity, and these two molecules can act modulating the antitumor immunity. The vitamin D also showed a significant compound with purinergic system modulation, since it is capable of increasing CD73 expression and controlling the adenosine amount, which seems to play a great role in cutaneous melanoma. Source: The authors (2022).*

#### *The Potential of the Purinergic System as a Therapeutic Target of Natural Compounds… DOI: http://dx.doi.org/10.5772/intechopen.105457*

hydrolysis of ADP in platelets of the animals treated compared with untreated. In the lymphocytes of the animals with the disease that received tannic acid, this polyphenol decreased the hydrolysis of ATP and ADP and the degradation of adenosine in relation to the group with the disease that did not receive the substance. When comparing the levels of ectonucleotidases in control mice, those with glioblastoma and those with glioblastoma treated with tannic acid, it was observed that the substance was able to maintain levels similar to those in mice without the disease. Bearing in mind that the purinergic system is able to modulate tumor progression, the aggressiveness of this type of cancer, and the results obtained in the study in question, tannic acid can be considered a promising agent for the treatment of cancer [89].

Regarding purinergic system modulation and the antitumor effect on cutaneous melanoma, Silva et al. [5] proposed the hypothesis that rosmarinic acid, a polyphenol with antitumor effect, would be able to modulate purinergic signaling and prevent tumor progression and metastasis by two-way means: by blocking the P2X7 receptor or by antagonizing the P2Y12 receptor. Interestingly, a paper that focused on Salvia *yunnanensis* extract, which contains rosmarinic acid in its composition, proved the inhibition ADP-induced of rabbit platelet aggregation by binding rosmarinic acid with P2Y12R [90].

Coffee (*Coffea arabica*) and green tea (*Camellia sinensis*) derivatives such as caffeine (1,3,7 trymetylxantine), caffeic acid (3,4-dihydroxycinnamic acid) and chlorogenic acid (3-O-caffeoylquinic acid) have shown promising effects in degenerative and cardiovascular diseases, in which they have been shown to modulate inflammation and purinergic signaling, mainly through the P1 family receptors that are closely related to tumor immunity, being strong candidates for the treatment of CM [91, 92]. To confirm the previous data, caffeic acid together with the antineoplastic dacarbazine decreased the viability of SK-Mel-28 metastatic cutaneous melanoma cells [93].

Quercetin, an abundant flavonoid in plants, also demonstrated antitumor activity in cell lines of bladder cancer, glioblastoma, and hepatocarcinoma and inhibited the activity and expression of ecto-5′- NT/CD73, leaving less Ado available in the TME, consequently preventing immunosuppression [94–96].

Apigenin (4′,5,7-trihydroxyflavone) is a flavonoid present in significant amounts in parsley, onion, celery, orange, chamomile, oregano, and basil that has shown a beneficial effect in diseases such as cancer, Alzheimer's, diabetes, and depression [97, 98]. Cutaneous melanoma cells (A375) were treated with these substance and, in addition to the decrease in cell viability, they had an increase in ATPase activity and a concomitant reduction in the ATP/ADP ratio related to the apoptotic process of cancer cells, demonstrating that the present substance has an effect antitumor in addition to acting in purinergic system [99].

The carcinogenesis can be initiated by the overproduction of reactive oxygen species (ROS), since the antioxidant defenses cannot neutralize these molecules. From this, the antioxidant compounds seem to be important against tumor formation, such as the phenolic acids, which can prevent DNA alterations and genome instability. The literature has shown that rosmarinic acid is an example of a powerful antioxidant for the protection of the DNA against UV and H2O2 [100].

Vitamin D, in turn, is a fat-soluble vitamin, in which its deficiency is closely related to carcinogenesis [101]. This vitamin is available in two forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol), where it can be obtained through the diet, but 90% of the daily needs are produced by the skin itself and later must be hydroxylated by the liver and kidneys, in order to obtain 1,25-dihydroxyvitamin D, the metabolically active form of vitamin D [102]. In metastatic melanoma

cells, SK-Mel-28-treated with 1,25-dihydroxyvitamin D, there was decrease in cell viability, as well as the expression and activity of the CD73 enzyme and the levels of Ado, which has a suppressive function of tumor immunity and is essential for progression tumor, becoming a promising candidate for the adjuvant treatment of cutaneous melanoma [103].

#### **5. Conclusion**

As evidenced, cutaneous melanoma is a malignant neoplasm of great medical importance due to high rates of resistance to treatments and relapses, and for this reason, it is necessary to search for new and effective pharmacological therapies. In this context, the potential of some compounds in modulations of this pathway signaling, such as rosmarinic acid, resveratrol, tannic acid, as well as vitamin D, has been elucidated in this study. Of course, more research needs to be done to open up new horizons in the treatment of melanoma by the purinergic signaling, but the discovery of new ways to improve the anticancer pharmacological perspective has already begun.

### **Funding**

MDB acknowledges grant support by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, proj. No 310606/2021–7) and Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC, proj. No 2021TR1543). GBS, DM, MM, and MDB also thank Fundo de Apoio à Manutenção e ao Desenvolvimento da Educação Superior (FUMDES/UNIEDU) for the graduate scholarships.

### **Conflict of interest**

The authors declare that there is no conflict of interest.

### **Author details**

Gilnei Bruno da Silva, Daiane Manica, Marcelo Moreno and Margarete Dulce Bagatini\* Graduate Program in Biomedical Sciences, Federal University of Fronteira Sul, Chapecó, SC, Brazil

\*Address all correspondence to: margaretebagatini@yahoo.com.br

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Potential of the Purinergic System as a Therapeutic Target of Natural Compounds… DOI: http://dx.doi.org/10.5772/intechopen.105457*

#### **References**

[1] Schadendorf D, et al. Melanoma. The Lancet. 2018;**392**(10151):971-984. DOI: 10.1016/S0140-6736(18)31559-9

[2] Paddock LE, et al. Skin selfexamination and long-term melanoma survival. Melanoma Research. 2016;**26**(4):401-408. DOI: 10.1097/ CMR.0000000000000255

[3] Siegel R, et al. Cancer statistics, 2013. CA: a Cancer Journal for Clinicians. 2013;**63**(1):11-30. DOI: 10.3322/caac.21166

[4] Wilson MA, Schuchter LM. Chemotherapy for melanoma. In: Kaufman HL, Mehnert JM, editors. Melanoma. Cancer Treatment and Research. Vol. v. 167. Cham: Springer International Publishing; 2016. pp. 209- 229. DOI: 10.1007/978-3-319-22539-5\_8

[5] da Silva GB, Yamauchi MA, Zanini D, Bagatini MD. Novel possibility for cutaneous melanoma treatment by means of rosmarinic acid action on purinergic signaling. Purinergic Signalling. 2022).;**18**:61-81. DOI: 10.1007/ s11302-021-09821-7

[6] Mânica A, Bagatini MD. Melanoma cutâneo e sistema purinérgico. In: Cardoso AM, Manfredi LH, Maciel SFV d O, editors. Sinalização Purinérgica: Implicações Fisiopatológicas. Chapecó: UFFS; 2021. pp. 156-171

[7] Miller AJ, Mihm MC. Melanoma. The New England Journal of Medicine. 2006;**355**(1):51-65

[8] Australian Institute of Health and Welfare 2021. Cancer in Australia 2021. Cancer series nº. 133. Cat. nº. CAN 144. Canberra: AIHW

[9] Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA: a Cancer Journal for Clinicians. 2022;**72**(1):7-33

[10] Garbe C, Keim U, Eigentler TK, Amaral T, Katalinic A, Holleczek B, et al. Time trends in incidence and mortality of cutaneous melanoma in Germany. Journal of the European Academy of Dermatology and Venereology. 2019;**33**(7):1272-1280

Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a Cancer Journal for Clinicians. 2021;**71**(3):209-249

[12] Olsen CM,

Whiteman DC. Clinical epidemiology of melanoma. In: Balch CM, Atkins MB, Garbe C, Gershenwald JE, Halpern AC, Kirkwood JM, et al., editors. Cutaneous Melanoma. Cham: Springer International Publishing; 2020. pp. 425-449

[13] INCA - Instituto Nacional do Câncer (Brazil). Cancer of Skin Melanoma. Brasília, DF: Instituto Nacional do Câncer; 2021. Available from: https:// www.inca.gov.br/tipos-de-cancer/cancerde-pele-melanoma Accessed: March 2021

[14] Moreno M, Schmidt JC, Grosbelli L, Dassi M, Mierzwa RV. Análise de prevalência e mortalidade associada ao melanoma cutâneo em pacientes atendidos em centro de referência no Oeste do estado de Santa Catarina, Brasil, de 2002 a 2016. Rev Ciênc EM SAÚDE. 2020;**10**(4):109-116

[15] Yang AS, Chapman PB. The history and future of chemotherapy for melanoma. Hematology/

<sup>[11]</sup> Sung H, Ferlay J,

Oncology Clinics of North America. 2009;**23**(3):583-597

[16] Chabner BA, Roberts TG. Chemotherapy and the war on cancer. Nature Reviews. Cancer. 2005;**5**(1):65-72

[17] DeVita VT, Chu E. A history of cancer chemotherapy. Cancer Research. 2008;**68**(21):8643-8653

[18] Finn OJ. Cancer immunology. The New England Journal of Medicine. 2008;**358**(25):2704-2715

[19] Flaherty KT, Robert C, Hersey P, Nathan P, Garbe C, Milhem M, et al. Improved survival with MEK inhibition in BRAF-mutated melanoma. The New England Journal of Medicine. 2012;**367**(2):107-114

[20] Colombino M, Capone M, Lissia A, Cossu A, Rubino C, De Giorgi V, et al. BRAF/NRAS mutation frequencies among primary Tumors and metastases in patients with melanoma. Journal of Clinical Oncology. 2012;**30**(20):2522-2529

[21] Kwak EL, Clark JW, Chabner B. Targeted agents: The rules of combination: Table 1. Clinical Cancer Research. 2007;**13**(18):5232-5237

[22] Leach DR, Krummel MF, Allison JP. Enhancement of antitumor immunity by CTLA-4 blockade. Science. 1996;**271**(5256):1734-1736

[23] Nakamura Y. Biomarkers for immune checkpoint inhibitor-mediated tumor response and adverse events. Frontiers in Medicine. 2019;**6**:119

[24] Orloff M, Weight R, Valsecchi ME, Sato T. Immune check point inhibitors combination in melanoma: Worth the toxicity? Reviews on Recent Clinical Trials. 2016;**11**(2):81-86

[25] Havel JJ, Chowell D, Chan TA. The evolving landscape of biomarkers for checkpoint inhibitor immunotherapy. Nature Reviews. Cancer. 2019;**19**(3): 133-150

[26] Burnstock G, et al. Cellular distribution and function of P2 receptor subtypes in different systems. International Review of Cytology. 2004;**240**:31-304. DOI: 10.1016/ S0074-7696(04)40002-3

[27] Atkinson B, et al. Ecto-nucleotidases of the CD39/NTPDase family modulate platelet activation and thrombus formation: Potential as therapeutic targets. Blood Cells, Molecules, and Diseases. 2006;**36**:217-222. DOI: 10.1016/j.bcmd.2005.12.025

[28] Burnstock G, et al. Purinergic Signaling, and the Nervous System. Berlin, Heidelberg: Springer; 2012. DOI: 10.1007/978-3-642-28863-0

[29] Visovatti SH, et al. Increased CD39 nucleotidase activity on microparticles from patients with idiopathic pulmonary arterial hypertension. PLoS One. 2012;**7**:408-429. DOI: 10.1371/journal. pone.0040829

[30] Bagatini MD, et al. The impact of purinergic system enzymes on noncommunicable, neurological, and degenerative diseases. Journal of Immunology Research. 2018;**2018**:1-21. DOI: 10.1155/2018/4892473

[31] Bartoli F, et al. Purinergic signaling and related biomarkers in depression. Brain Sciences. 2020;**10**(3):1-12

[32] Zimmermann H. Cellular function and molecular structure of ectonucleotidases. Purinergic Signalling. 2012;**8**:437-502. DOI: 10.1007/ s11302-012-9309-4

[33] Yegutkin GG, et al. Nucleotide-and nucleoside-converting ectoenzymes:

*The Potential of the Purinergic System as a Therapeutic Target of Natural Compounds… DOI: http://dx.doi.org/10.5772/intechopen.105457*

Important modulators of purinergic signalling cascade. Biochimica et Biophysica Acta – Molecular Cell Research. 2008;**1783**:673-694. DOI: 10.1016/j.bbamcr.2008.01.024

[34] Schetinger MRC, et al. NTPDase and 5-nucleotidase activities in physiological and disease conditions: New perspectives for human health. BioFactors. 2007;**31**:77-98. DOI: 10.1002/ biof.5520310205

[35] Zimmermann H. Ectonucleotidases in the nervous system. Novartis Foundation Symposium. 2006;**276**:113-128

[36] Zanini D, et al. Ectoenzymes and cholinesterase activity and biomarkers of oxidative stress in patients with lung cancer. Molecular and Cellular Biochemistry. 2013;**374**(1-2):137-148. DOI: 10.1007/s11010-012-1513-6

[37] Zanini D, et al. ADA activity is decreased in lymphocytes from patients with advanced stage of lung ca9ncer. Medical Oncology. 2019.;**36**:78. DOI: 10.1007/s12032-019-1301-1

[38] Ledderose C, et al. Cutting off the power: Inhibition of leukemia cell growth by pausing basal ATP release and P2X receptor signaling? Purinergic Signal. 2016;**12**(3):439-451. DOI: 10.1007/ s11302-016-9510-y

[39] Mânica A, et al. The signaling effects of ATP on melanoma-like skin cancer. Cellular Signalling. 2019;**59**:122-130. DOI: 10.1016/j.cellsig.2019.03.021

[40] Hu L-P, et al. Targeting purinergic receptor P2Y2 prevents the growth of pancreatic ductal adenocarcinoma by inhibiting cancer cell glycolysis. Clinical Cancer Research. 2019;**25**(4):1318-1330. DOI: 10.1158/1078-0432.CCR-18-2297 [41] Hevia MJ, et al. Differential effects of purinergic Signaling in gastric cancerderived cells through P2Y and P2X receptors. Frontiers in Pharmacology. 2019;**10**:612. DOI: 10.3389/fphar.2019. 00612

[42] Burnstock G. Short- and longterm (trophic) purinergic signalling. Philosophical Transactions of the Royal Society B: Biological Sciences. 2016.;**371**:1700. DOI: 10.1098/ rstb.2015.0422

[43] Stagg J, Smyth MJ. Extracellular adenosine triphosphate and adenosine in cancer. Oncogene. 2010;**29**(39): 5346-5358. DOI: 10.1038/onc.2010.292

[44] Burnstock G. Blood cells: An historical account of the roles of purinergic signalling. Purinergic Signalling. 2015;**11**(4):411-434. DOI: 10.1007/s11302-015-9462-7

[45] Di Virgilio F, et al. Extracellular purines, purinergic receptors and tumor growth. Oncogene. 2017.;**36**(3):293-303. DOI: 10.1038/onc.2016.206

[46] Ghiringhelli F, et al. Activation of the NLRP3 inflammasome in dendritic cells induces IL-1β–dependent adaptive immunity against tumors. Nature Medicine. 2009;**15**(10):1170-1178. DOI: 10.1038/nm.2028

[47] Bian S, Sun X, Bai A, Zhang C, Li L, Enjyoji K, et al. P2X7 integrates PI3K/AKT and AMPK-PRAS40-mTOR signaling pathways to mediate tumor cell death. PLoS One. 2013;**8**:60184. DOI: 10.1371/journal.pone.0060184

[48] Di Virgilio F. Purines, purinergic receptors, and cancer. Cancer Research. 2012;**72**(21):5441-5447. DOI: 10.1158/0008-5472.CAN-12-1600

[49] Feng L, et al. Vascular CD39/ENTPD1 directly promotes tumor cell growth

by scavenging extracellular adenosine triphosphate. Neoplasia. 2011;**13**(3):206- 216. DOI: 10.1593/neo.101332

[50] Mânica A, et al. High levels of extracellular ATP lead to chronic inflammatory response in melanoma patients. Journal of Cellular Biochemistry. 2018;**119**(5):3980-3988. DOI: 10.1002/jcb.26551

[51] Whintton B, et al. Vacuolar ATPase as a potential therapeutic target and mediator of treatment resistance in cancer. Cancer Medicine, 7, 8, p. 3800- 3811, 2018. DOI: 10.1002/cam4.1594

[52] Di Virgilio F, et al. Extracellular ATP and P2 purinergic signalling in the tumour microenvironment. Nature Reviews Cancer. 2018;**18**(10):601-618. DOI: 10.1038/s41568-018-0037-0

[53] Pasquali S, et al. Systemic treatments for metastatic cutaneous melanoma. The Cochrane Database of Systematic Reviews. 2018;**2**(2):CD011123. DOI: 10.1002/14651858.CD011123.pub2

[54] Pegoraro A, et al. P2X7 promotes metastatic spreading and triggers release of miRNA-containing exosomes and microvesicles from melanoma cells. Cell Death & Disease. 2021;**12**:1088. DOI: 10.1038/s41419-021-04378-0

[55] Hattori F, et al. Feasibility study of B16 melanoma therapy using oxidized ATP to target purinergic receptor P2X7. European Journal of Pharmacology. 2012;**625**:20-26. DOI: 10.1016/j. ejphar.2012.09.001

[56] White N, et al. Human melanomas express functional P2X7 receptors. Cell and Tissue Research. 2005;**321**:411-418. DOI: 10.1007/s00441-005-1149-x

[57] Romagnani A, et al. P2X7 receptor activity limits accumulation of T cells within tumors. Cancer Research. 2020;**80**(18):3906-3919. DOI: 10.1158/0008-5472.CAN-19-3807

[58] White N, et al. P2Y purinergic receptors regulate the growth of human melanomas. Cancer Letters. 2005;**224**(1):81-91. DOI: 10.1016/j. canlet.2004.11.027

[59] Gebremeskel S, et al. The reversible P2Y12 inhibitor ticagrelor inhibits metastasis and improves survival in mouse models of cancer: P2Y12 inhibitor ticagrelor inhibits metastasis. International Journal of Cancer. 2015.;**136**(1):234-240. DOI: 10.1002/ ijc.28947

[60] Kamiyama M, et al. ASK1 facilitates tumor metastasis through phosphorylation of an ADP receptor P2Y12 in platelets. Cell Death and Differentiation. 2017;**24**:2066-2076. DOI: 10.1038/cdd.2017.114

[61] Ruzsnavszky O, et al. UV-B induced alteration in purinergic receptors and signaling on HaCaT keratinocytes. Journal of Photochemistry and Photobiology B: Biology. 2011;**105**(1):113-118. DOI: 10.1016/j. jphotobiol.2011.07.009

[62] Merighi S, et al. Adenosine receptors as mediators of both cell proliferation and cell death of cultured human melanoma cells. Journal of Investigative Dermatology. 2002;**119**(4):923-933. DOI: 10.1046/j.1523-1747.2002.00111.x

[63] Cekic C, et al. Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment. Cancer Research. 2014;**74**(24):7250-7259. DOI: 10.1158/0008-5472.CAN-13-3583

[64] Fishman P, et al. A3 adenosine receptor as a target for cancer therapy. *The Potential of the Purinergic System as a Therapeutic Target of Natural Compounds… DOI: http://dx.doi.org/10.5772/intechopen.105457*

Anti-Cancer Drugs. 2002;**13**(5):437-443. DOI: 10.1097/00001813-200206000- 00001

[65] Koszałka P, et al. Specific activation of A3, A2A and A1 adenosine receptors in CD73-knockout mice affects B16F10 melanoma growth, neovascularization, angiogenesis and macrophage infiltration. PLoS One. 2016.;**11**(3):e0151420. DOI: 10.1371/ journal.pone.0151420

[66] Merighi S, et al. A2B and A3 adenosine receptors modulate vascular endothelial growth factor and interleukin-8 expression in human melanoma cells treated with etoposide and doxorubicin. Neoplasia. 2009;**11**:1064-1073. DOI: 10.1593/ neo.09768

[67] Aliagas E, et al. High expression of ecto-nucleotidases CD39 and CD73 in human endometrial tumors. Mediators of Inflammation. 2014;**2014**:509027. DOI: 10.1155/2014/509027

[68] Cappellari AR, et al. Characterization of ectonucleotidases in human medulloblastoma cell lines: Ecto-5'NT/CD73 in metastasis as potential prognostic factor. PLoS One. 2012;**7**:e47468. DOI: 10.1371/journal. pone.0047468

[69] Longhi MS, et al. Biological functions of ecto-enzymes in regulating extracellular adenosine levels in neoplastic and inflammatory disease states. Journal of Molecular Medicine (Berlin, Germany). 2013;**91**:165-172. DOI: 10.1007/s00109-012-0991-z

[70] Jiang T, et al. Comprehensive evaluation of NT5E/CD73 expression and its prognostic significance in distinct types of cancers. BMC Cancer. 2018;**18**(1):267. DOI: 10.1186/ s12885-018-4073-7

[71] Sadej R, et al. Expression of ecto-5′-nucleotidase (eN, CD73) in cell lines from various stages of human melanoma. Melanoma Research. 2006;**16**(3):213-222. DOI: 10.1097/01. cmr.0000215030.69823.11

[72] I. Monteiro, et al. CD73 expression and clinical significance in human metastatic melanoma. Oncotarget, 9, 42, p. 26659-26669. 2018. DOI: 10.18632/ oncotarget.25426.

[73] Forte G, et al. Inhibition of CD73 improves B cell-mediated anti-tumor immunity in a mouse model of melanoma. The Journal of Immunology. 2012;**189**(5):2226-2233. DOI: 10.4049/ jimmunol.1200744

[74] Sadej R, et al. Dual, enzymatic and non-enzymatic, function of ecto-5′ nucleotidase (eN, CD73) in migration and invasion of A375 melanoma cells. Acta Biochimica Polonica. 2012;**59**(4):647-652

[75] Petric R, Braicu C, Raduly L, Dragos N, Dumitrascu D, Berindan-Negoe I, et al. Phytochemicals modulate carcinogenic signaling pathways in breast and hormone-related cancers. Onco Targets and Therapy. 2015;**8**:2053. DOI: 10.2147/OTT.S83597

[76] Niedzwiecki A,

Roomi M, Kalinovsky T, Rath M. Anticancer efficacy of polyphenols and their combinations. Nutrients. 2016;**8**(9):552. DOI: 10.3390/nu8090552

[77] Kozikowski AP, Tückmantel W, Böttcher G, Romanczyk LJ. Studies in polyphenol chemistry and bioactivity. 4. <sup>1</sup> Synthesis of trimeric, tetrameric, pentameric, and higher oligomeric epicatechin-derived procyanidins having all-4β,8-interflavan connectivity and their inhibition of cancer cell growth through cell cycle arrest <sup>1</sup> . The Journal

of Organic Chemistry. 2003;**68**(5):1641- 1658. DOI: 10.1021/jo020393f

[78] Santos-Buelga C, González-Paramás AM, Oludemi T, Ayuda-Durán B, González-Manzano S. Plant phenolics as functional food ingredients. Advances in Food and Nutrition Research. 2019;**90**:183-257. DOI: 10.1016/bs.afnr.2019.02.012

[79] Manach C, Scalbert A, Morand C, Rémésy C, Jiménez L. Polyphenols: Food sources and bioavailability. The American Journal of Clinical Nutrition. 2004;**79**(5):727-747. DOI: 10.1093/ ajcn/79.5.727

[80] Lakenbrink C, Lapczynski S, Maiwald B, Engelhardt UH. Flavonoids and other polyphenols in consumer brews of tea and other caffeinated beverages. Journal of Agricultural and Food Chemistry. 2000;**48**(7):2848-2852. DOI: 10.1021/jf9908042

[81] Rodríguez-García C, Sánchez-Quesada C, Gaforio JJ. Dietary flavonoids as cancer chemopreventive agents: An updated review of human studies. Antioxidants. 2019;**8**(5):137. DOI: 10.3390/antiox8050137

[82] Kopustinskiene DM, Jakstas V, Savickas A, Bernatoniene J. Flavonoids as anticancer agents. Nutrients. 2020;**12**(2):457. DOI: 10.3390/nu12020457

[83] Bottari NB, Pillat MM, Schetinger MRC, Reichert KP, Machado V, Assmann CE, et al. Resveratrol-mediated reversal of changes in purinergic signaling and immune response induced by Toxoplasma gondii infection of neural progenitor cells. Purinergic Signalling. 2019;**15**(1):77-84. DOI: 10.1007/ s11302-018-9634-3

[84] Fracasso M, Reichert K, Bottari NB, da Silva AD, Schetinger MRC, Monteiro SG, et al. Involvement of ectonucleotidases and purinergic receptor expression during acute Chagas disease in the cortex of mice treated with resveratrol and benznidazole. Purinergic Signalling. 2021;**17**(3):493-502. DOI: 10.1007/ s11302-021-09803-9

[85] Marinheiro D, Ferreira B, Oskoei P, Oliveira H, Daniel-da-Silva A. Encapsulation and enhanced release of resveratrol from mesoporous silica nanoparticles for melanoma therapy. Materials. 2021;**14**(6):1382. DOI: 10.3390/ma14061382

[86] Sánchez-Melgar A, Muñoz-López S, Albasanz JL, Martín M. Antitumoral action of resveratrol through adenosinergic signaling in C6 glioma cells. Frontiers in Neuroscience. 2021;**15**:702817. DOI: 10.3389/ fnins.2021.702817

[87] Bridgeman CJ, Nguyen T-U, Kishore V. Anticancer efficacy of tannic acid is dependent on the stiffness of the underlying matrix. Journal of Biomaterials Science, Polymer Edition. 2018;**29**(4):412-427. DOI: 10.1080/09205063.2017.1421349

[88] Nagesh PKB, Chowdhury P, Hatami E, Jain S, Dan N, Kashyap VK, et al. Tannic acid inhibits lipid metabolism and induce ROS in prostate cancer cells. Scientific Reports. 2020;**10**(1):980. DOI: 10.1038/ s41598-020-57932-9

[89] Bona NP, Soares MSP, Pedra NS, Spohr L, da Silva dos Santos F, de Farias AS, et al. Tannic acid attenuates peripheral and brain changes in a preclinical rat model of glioblastoma by modulating oxidative stress and purinergic signaling. Neurochemical Research [Internet]. 2022;**47**:1541-1552. DOI: 10.1007/s11064-022-03547-7

*The Potential of the Purinergic System as a Therapeutic Target of Natural Compounds… DOI: http://dx.doi.org/10.5772/intechopen.105457*

[90] Li Y, et al. Screening for the antiplatelet aggregation quality markers of Salvia yunnanensis based on an integrated approach. Journal of Pharmaceutical and Biomedical Analysis. 2020;**188**:113383. DOI: 10.1016/j. jpba.2020.113383

[91] Stefanello N, Spanevello RM, Passamonti S, Porciúncula L, Bonan CD, Olabiyi AA, et al. Coffee, caffeine, chlorogenic acid, and the purinergic system. Food and Chemical Toxicology. 2019;**123**:298-313. DOI: 10.1016/j. fct.2018.10.005

[92] Castro MFV, Stefanello N, Assmann CE, Baldissarelli J, Bagatini MD, da Silva AD, et al. Modulatory effects of caffeic acid on purinergic and cholinergic systems and oxi-inflammatory parameters of streptozotocininduced diabetic rats. Life Sciences. 2021;**277**:119421. DOI: 10.1016/j. lfs.2021.119421

[93] Pelinson LP, Assmann CE, Palma TV, da Cruz IBM, Pillat MM, Mânica A, et al. Antiproliferative and apoptotic effects of caffeic acid on SK-Mel-28 human melanoma cancer cells. Molecular Biology Reports. 2019;**46**(2):2085-2092. DOI: 10.1007/ s11033-019-04658-1

[94] Braganhol E, Tamajusuku ASK, Bernardi A, Wink MR, Battastini AMO. Ecto-5′-nucleotidase/CD73 inhibition by quercetin in the human U138MG glioma cell line. Biochimica et Biophysica Acta (BBA) - General Subjects. 2007;**1770**(9):1352-1359. DOI: 10.1016/j. bbagen.2007.06.003

[95] Rockenbach L, Bavaresco L, Fernandes Farias P, Cappellari AR, Barrios CH, Bueno Morrone F, et al. Alterations in the extracellular catabolism of nucleotides are involved in the antiproliferative effect of quercetin in human bladder

cancer T24 cells. Urologic Oncology: Seminars and Original Investigations. 2013;**31**(7):1204-1211. DOI: 10.1016/j. urolonc.2011.10.009

[96] Abruzzese V, Matera I, Martinelli F, Carmosino M, Koshal P, Milella L, et al. Effect of quercetin on ABCC6 transporter: Implication in HepG2 migration. International Journal of Molecular Sciences. 2021;**22**(8):3871. DOI: 10.3390/ijms22083871

[97] Hostetler GL, Ralston RA, Schwartz SJ. Flavones: Food sources, bioavailability, metabolism, and bioactivity. Advances in Nutrition. 2017;**8**(3):423-435. DOI: 10.3945/ an.116.012948

[98] Salehi B, Venditti A, Sharifi-Rad M, Kręgiel D, Sharifi-Rad J, Durazzo A, et al. The therapeutic potential of apigenin. International Journal of Molecular Sciences. 2019;**20**(6):1305. DOI: 10.3390/ ijms20061305

[99] Das S, Das J, Samadder A, Boujedaini N, Khuda-Bukhsh AR. Apigenin-induced apoptosis in A375 and A549 cells through selective action and dysfunction of mitochondria. Experimental Biology and Medicine (Maywood, N.J.). 2012;**237**(12):1433-1448. DOI: 10.1258/ ebm.2012.012148

[100] Sevgi K, et al. Antioxidant and DNA damage protection potentials of selected phenolic acids. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association. 2015;**77**:12-21. DOI: 10.1016/j. fct.2014.12.006

[101] Holick MF. Vitamin D: Its role in cancer prevention and treatment. Progress in Biophysics and Molecular Biology. 2006;**92**(1):49-59. DOI: 10.1016/j.pbiomolbio.2006.02.014 [102] Lips P. Vitamin D physiology. Progress in Biophysics and Molecular Biology. 2006;**92**(1):4-8. DOI: 10.1016/j. pbiomolbio.2006.02.016

[103] Bagatini MD,

Bertolin K, Bridi A, Pelinson LP, da Silva Rosa Bonadiman B, Pillat MM, et al. 1α, 25-Dihydroxyvitamin D3 alters ectonucleotidase expression and activity in human cutaneous melanoma cells. Journal of Cellular Biochemistry. 2019;**120**(6):9992-10000. DOI: 10.1002/ jcb.28281

### *Edited by Margarete Dulce Bagatini*

Characterized as a common signaling pathway between cells, the purinergic system is capable of modulating physiological and biochemical processes. Composed of signaling molecules, regulatory enzymes, and specific receptors, this organization can modulate several basal pathways of the organism. It is understood that purinergic signaling is present in all aspects of immunity and inflammation and studies show that extracellular ATP and its adenosine metabolite are the main mediators of response. These occur since most immune cells express P2 and P1 receptors, which are sensitive to the ATP and adenosine molecules, respectively. This book describes the purinergic system and its correlation with the health and disease process.

### *Miroslav Blumenberg, Biochemistry Series Editor*

Published in London, UK © 2022 IntechOpen © monsitj / iStock

Purinergic System

IntechOpen Series

Biochemistry, Volume 36

Purinergic System

*Edited by Margarete Dulce Bagatini*