P2 purinergic receptors regulate the progression of colorectal cancer

Abstract

More and more studies have revealed that P2 purinergic receptors play a key role in the progression of colorectal cancer (CRC). P2X and P2Y purinergic receptors can be used as promoters and regulators of CRC and play a dual role in the progression of CRC. CRC microenvironment is rich in ATP and its cleavage products (ADP, AMP, Ado), which act as activators of P2X and P2Y purinergic receptors. The activation of P2X and P2Y purinergic receptors regulates the progression of CRC mainly by regulating the function of immune cells and mediating different signal pathways. In this paper, we focus on the specific mechanisms and functional roles of P2X7, P2Y12, and P2Y2 receptors in the growth and progression of CRC. The antagonistic effects of these selective antagonists of P2X purinergic receptors on the growth, invasion, and metastasis of CRC were further discussed. Moreover, different studies have reported that P2X7 receptor can be used as an effective predictor of patients with CRC. All these indicate that P2 purinergic receptors are a key regulator of CRC. Therefore, antagonizing P2 purinergic receptors may be an innovative treatment for CRC.

Introduction

Colorectal cancer (CRC) is a common malignant tumor in the digestive system. Its pathological mechanism is complex and involves many factors, which is closely related to the interaction between the tumor itself and the tumor microenvironment [1, 2]. Currently, the treatment of CRC is still a difficult problem to break through, although the combined treatment of CRC, such as chemotherapy, radiotherapy, surgery, and immunotherapy, has improved the survival rate and quality of life of patients. However, the growth, recurrence, metastasis, and drug resistance of CRC are still the main causes of death. Especially for patients with advanced distant metastasis, the best time for surgical treatment has been lost, and the application of molecular targeted therapy can improve the survival rate of patients. Therefore, it is particularly important to find and explore the molecular basis and key regulatory factors in the pathogenesis of CRC, and to find effective molecular targets for effective targeted therapy.

Tumor cells and other cells invaded by tumors in the CRC microenvironment can release purine nucleotides and nucleosides, such as ATP, ADP, AMP, and adenosine, to the outside of the cells, which can be produced by the tumor microenvironment [3, 4]. There, these substances regulate the progression of CRC, including growth or apoptosis, by acting on P2X and P2Y purinergic receptors on tumor and non-tumor cell membranes [5, 6]. At present, ion channel P2X purinergic receptors can be divided into 1–7 subtypes, especially P2X7 receptor has received extensive attention in CRC. ATP in tumor microenvironment activates these P2X purinergic receptors by opening non-selective positive ion channels (calcium, sodium, and potassium) on the cell membrane, and by activating different intracellular signal pathways to regulate intracellular molecular metabolism and thus regulating the progress of tumor cells [6–8]. Currently, G protein coupled P2Y purinergic receptors can be divided into 8 subtypes (1 2, 4, 6, 11–14). In the progression of metastatic cancer, G protein-coupled P2Y purinergic receptors are considered to be promising targets for cancer therapy, especially P2Y12 and P2Y2 receptors in CRC [6] (Table 1).

Table 1.

Study on the role of P2 purinergic receptor in CRC

Purinergic receptor subtype	Expression of CRC cells	Expression in CRC tissues	In vitro action	In vivo action	Prognosis of clinical patients	Ref
P2X7 receptor		High expression		P2X7 receptor (+ / +) mice showed more ulcers, tumors, and larger wall thickness and increased immune cell accumulation and pro-inflammatory cytokines production		10
P2X7 receptor	High expression		P2X7 receptor antagonist A438079 can inhibit the proliferation, invasion, and migration of HCT-116 and SW620 cells	A438079 inhibits the growth of CRC in nude mice		35
P2X7 receptor	High expression		P2X7 receptor activation promotes the proliferation, migration, and invasion of CRC cells. Knockdown of P2X7 receptor gene expression or the use of antagonists A438079 and AZD9056 can inhibit the proliferation and migration of CRC cells			36
P2X7 receptor	High expression	High or low expression			Patients with high expression of P2X7 receptor have relatively short survival time, higher serum carcinoembryonic antigen levels, and more advanced tumors	37
P2X7 receptor	High expression	High expression	Activation of P2X7 receptor promotes the proliferation of CRC cells		The high expression of P2X7 receptor was significantly correlated with tumor size, lymph node metastasis, and TNM stage. Univariate and multivariate COX regression analysis showed that high expression of P2X7 receptor could be used as an independent prognostic factor affecting overall survival	38
P2X7 receptor		High or low expression			Overexpression of P2X7 receptor was related to TILs, depth of invasion, distant metastasis, and staging. Multivariate COX regression analysis showed that overexpression of P2X7 receptor was significantly correlated with overall survival rate	39
P2Y2 receptor	High expression		Inhibition of P2Y2 receptor completely eliminated the increase of intracellular calcium in all CRC cell lines after ATP exposure			40
P2X7 receptor	High expression		P2X7 receptor activation promotes the migration and invasion of colon cancer cells by activating STAT3 signal. P2X7 receptor antagonists A438079 and AZD9056 or P2X 7 siRNA could inhibit the migration and invasion of CRC cells induced by ATP	P2X7 receptor activation induced by ATP promotes tumor growth in vivo		49
P2Y2 receptor			P2Y2 receptor agonists can induce apoptosis and inhibit proliferation of CRC cells Colo320 or HT29 in a time-dependent manner			53
P2X7 receptor	High expression	High expression	Overexpression of P2X7 receptor increases the expression of CD31 and VEGF and promotes angiogenesis. Transplanted tumor cells with high expression of P2X7 receptors can stimulate cytokines to recruit tumor-associated macrophages, thus promoting tumor growth	P2X7 receptor promotes the growth and angiogenesis of CRC in vivo		69
P2X7 receptor	High expression		The increase of membrane fluidity induced by high temperature enhanced the function of P2X7 receptor and the opening of pores, regulated downstream AKT/PRAS40/mTOR signal events, and had a killing effect on MCA38 colon cancer cells			71
P2X7 receptor				In P2X7 receptor gene deficient mice, the tumor growth and metastasis and diffusion rate were significantly accelerated, the release of IL-1β and VEGF was significantly decreased, and inflammatory cell infiltration was almost completely eliminated		72
P2Y2 receptor	High expression		P2Y2 activates Src, which in turn phosphorylates p38, resulting in overexpression of COX-2, which induces resistance to apoptosis in HT-29 cells			81
P2Y12 receptor	High expression		The overexpression of P2Y12 receptor promotes the proliferation of tumor	Inhibition of P2Y12 receptor expression has smaller primary tumors and less metastasis		87
P2Y12 receptor		High expression	Ticagrel, a P2Y12 receptor antagonist, significantly inhibited platelet-platelet aggregation induced by CRC cells	Inhibition of P2Y12 receptor activity may reduce spontaneous platelet aggregation and activation in patients with metastatic cancer	The levels of spontaneous platelet aggregation and activation in cancer patients taking ticagrel were significantly lower than the baseline level	91
P2Y12 receptor	High expression		P2Y12 receptor antagonist ticagrel inhibited the activation of Ado and inhibited the EMT and migration of platelets and cancer cells Ticagrel could prevent the down-regulation of E-cadherin in HT29 cells co-cultured with platelets and inhibited the migration ability of HT29 cells			92

In addition, the immune cells in the tumor microenvironment are also the action points of P2 purinergic receptors. These P2 purinergic receptors can mediate the activation of immune cells, exert immunosuppression or immune escape to drive CRC to produce immune tolerance, thus reducing the therapeutic effect of drugs [8–10]. Interestingly, P2 purinergic receptor antagonists can antagonize these P2 purinergic receptor activities, improve these factors in the tumor microenvironment, and inhibit the growth, migration, and invasion of CRC [6, 10, 11]. Although the functional role of P2 purinergic receptors in different cancers has been affirmed and recognized by different studies, there is still a lack of understanding and insight into the molecular mechanism of P2 purinergic receptors regulating the development of CRC. Here, we focus on the inherent relationship of P2 purinergic receptors in CRC, which provides some valuable information for the prevention and treatment of CRC.

Tumor microenvironment is rich in ATP and its cleavage products

The growth of tumor cells depends on the microenvironment in which they exist, which includes a variety of components, such as immune cells, ATP, stromal cells, exosomes, and growth factors. These factors can interact with each other to induce the migration and invasion of tumor cells [12, 13]. On the one hand, the growth and invasive behavior of tumor depend partly on the specific environment of tumor growth. On the other hand, this environment can inhibit the function of infiltrating inflammatory cells to facilitate tumor growth [12, 14]. Tumor cells can change this microenvironment by autocrine or paracrine to better adapt to their own progress [14]. ATP in tumor microenvironment plays a central role in cancer metabolism and progression. It is not only the energy substance of tumor cells, but also the signal transduction molecules involved in tumor cell behavior [15, 16]. ATP synthase-related peptides promote the construction of ATP synthase by interacting with α and γ subunits (ATP5A and ATP5C), increase the activity of ATP synthase and the oxygen consumption rate of mitochondria, and thus promote the proliferation of CRC cells [17]. The concentration of ATP in tumor was 10 (4) times higher than that in normal tissue. Phagocytosis of human A549 lung cancer cells internalizes extracellular ATP, reduces extracellular ATP concentration, and produces resistance to anticancer drugs [18]. This means that the resistance of cancer cells to ATP competitive anticancer drugs may be realized through the mechanism of ATP internalization.

Tumor cells can absorb extracellular ATP through phagocytosis of macrophages, thus increasing the level of intracellular ATP and improving its survival rate in drug therapy [19]. ATP internalization and increased intracellular ATP induce drug resistance in different types of tumors, thus revealing the role of tumor microenvironment rich in ATP in tumor drug resistance [19]. Other interesting studies have shown that the use of ATP responsive drug delivery, as a trigger for therapeutic drug delivery, can produce a more accurate drug delivery system and better drug release control and play a good role in anti-tumor [20, 21].

ATP and its cleavage products can be used as key signal molecules, which are closely related to cell proliferation, differentiation, and migration. Another important role of these substances in regulating the progress of tumor cells is that the immune cells in the microenvironment include macrophages, lymphocytes, and dendritic cells. The activity of these immune cells produces inflammatory response, regulates the immune microenvironment, and produces anti-tumor immunity and drug resistance [15, 22]. CD39 and CD73 can catalyze the decomposition of extracellular ATP into adenosine and the subsequent activation of different subtypes of adenosine receptors, resulting in anti-tumor or pro-tumor effects [4]. The concentrations of ATP and Ado are controlled by CD39 and CD73. Once accumulated in the tumor microenvironment, ATP can enhance the anti-tumor immune response, while Ado can weaken the anti-tumor immunity [4, 23]. Triggering the ATP-P2X7-NLRP3-IL18 axis can reduce the number of macrophages and enhance the T cell response function and the effectiveness of T cell metastasis in the tumor [24]. Tumor cells release more ATP through death and stress and convert their extracellular enzymes into Ado to create an adenosine-rich immunosuppressive microenvironment [4].

Adenosine is one of the most effective immunosuppressive factors, which can be regulated by binding to four adenosine receptors (A1R, A2AR, A2BR, and A3R) [25]. It has been found that A2AR induces immunosuppressive function by regulating cAMP signal. CD39/CD73/A2AR can be used as a new therapeutic target for regulating anti-tumor immunity [25]. Exosomes circTMEM181 from hepatocellular carcinoma promotes immunosuppression and anti-PD1 resistance by increasing the expression of CD39. Anti-PD1 resistance of hepatocytes can be saved by targeting CD39 on macrophages to inhibit ATP- adenosine pathway [26]. CD39 inhibitor POM-1 and anti-CD73 antibody inhibit adenosine production and T cell inhibition in myeloma and stromal cell co-culture in vitro [27]. AZD4635, an anti-CD73 and A2AR antagonist, blocks the adenosine pathway in vivo, activates T cells, increases interferon-γ production, and reduces tumor load [27]. Other interesting studies have revealed that nanoparticles carrying the extracellular nucleotidase inhibitor ARL67156 produce reactive oxygen species and promote immunogenic cell death induced by cancer therapy, including ATP release, while limit the degradation of ATP to adenosine to achieve a lasting anti-tumor immune response [28]. These studies have revealed the key role of ATP and its cleavage in tumor immunity in microenvironment, and the regulation of ATP/adenosine pathway may become a new method and strategy of anti-tumor immunotherapy.

Expression of P2 purinergic receptors in CRC and their clinical significance

Different studies have revealed the expression of P2X purinergic receptor subtypes P2X3, P2X5, P2X4, and P2X7 receptors in CRC [8, 29–31]. P2-specific mRNA expression can be detected in CRC cells and CRC tissues [32] However, the most studied P2X subtype is P2X7 receptor. P2X7 receptor is expressed in different types of CRC cell lines, such as SW620, HCT-116, LoVo and HT-29 [33–35]. P2X7 receptor agonists ATP and BzATP increased P2X7 receptor expression in SW620 and HCT-116 cells, while P2X7 receptor antagonists A438079 and AZD9056 decreased BzATP-induced P2X7 receptor expression [35]. The expression of P2X7 receptor was significantly up-regulated in metastatic CRC and metastatic CRC cell lines [36].

P2X7 receptor is highly expressed in patients with CRC, which is closely related to clinical prognosis, including lymph node metastasis, vascular invasion, and overall survival prognosis. It can be used as a marker for predicting CRC. The expression of P2X7 receptor was detected in 97 CRC specimens, normal colorectal tissues, and different CRC cell lines. The results showed that there were high P2X7 receptor and low P2X7 receptor populations in patients with CRC. Patients with high P2X7 receptor expression had relatively short survival time, higher serum carcinoembryonic antigen levels, and more advanced tumors [36]. High expression of P2X7 receptor was found in paraffin-embedded CRC tissues. High expression of P2X7 receptor was significantly correlated with tumor size, lymph node metastasis, and TNM stage [37]. Univariate and multivariate COX regression analysis showed that the high expression of P2X7 receptor could be used as an independent prognostic factor for overall survival in patients with CRC [37]. Another study yielded similar results by detecting the expression of P2X7 receptor and GLUT-1 in 196 cases of CRC. The overexpression of P2X7 receptor and GLUT-1 is related to TILs, depth of invasion, distant metastasis, and staging [38]. Multivariate COX regression analysis showed that overexpression of P2X7 receptor was significantly correlated with overall survival rate [38]. These studies have revealed the fact that P2X purinergic receptor (P2X7 receptor) may be a favorable predictive target for CRC and have important clinical significance for clinical prevention and treatment of CRC.

Different studies have shown that CRC cells express these P2Y subtypes of receptors (1, 2, 4, 6, 11–14) [6, 31, 39, 40]. For example, P2Y4 receptor positive reaction was detected by western blotting and immunohistochemistry in HT-29 cell line [41]. These P2Y purinergic receptors are strictly controlled by extracellular nucleotidase and kinase, and interact with nucleotides and nucleosides to change their availability and activity. P2Y1, P2Y12, and P2Y13 receptors are activated by ATP and ADP, P2Y2 and P2Y4 receptors are activated by UTP, P2Y6 receptor is activated by UDP, and P2Y14 receptor is activated by UDP-glucose [42]. These activators activate P2Y purinergic receptors to participate in the behavioral activity of CRC [43]. Another study identified the high expression pattern of P2Y receptors (P2Y2 and P2Y4 receptors) in CRC tissues. P2Y purinergic receptors (P2Y2 and P2Y4 receptors) can be detected in non-tumor tissues, but they are significantly overexpressed in CRC tissues [44]. These studies reveal that P2Y purinergic receptors play a certain role in CRC.

The role of P2 purinergic receptor-mediated signal pathway in CRC

As mentioned earlier, the tumor microenvironment is rich in ATP and its cleavage products, which act as activators of P2 purinergic receptors (P2X and P2Y). These substances activate P2 purinergic receptors to mediate different intracellular signal pathways to regulate the progression of CRC. Different studies have shown the activation of P2X7 receptor to regulate CRC progression by mediating different intracellular signal pathways [3, 45]. It is understood that P2X7 receptor activation mediates the opening of ion channels and mainly mediates calcium influx. Activation of P2X7 receptor mediates calcium signal to promote tumor progression. ATP promotes cell survival by regulating cytoplasmic [Ca²⁺] signal and Bcl-2/Bax ratio in A549 and H23 lung cancer cells [46]. ATP or BzATP significantly increased calcium influx in colon cancer cell lines LoVo and SW480, while P2X7 receptor antagonists A438079 and AZD9056 decreased ATP-induced calcium influx [47]. P2X7 receptor activation can also regulate the progress of intracellular signal transduction (such as AKT, ERK, STAT3, and mTOR) in the regulation of CRC. Studies have shown that P2X7 receptor activation promotes the growth and metastasis of colorectal cancer by activating PI3K/Akt/GSK-3β signal pathway [35]. Our previous studies have shown that ATP or BzATP activates P2X7 receptor and promotes the growth of LoVo and SW480 colon cancer cells by activating STAT3 signal pathway [47]. P2X7 receptor antagonist A438079 promotes apoptosis of HCT-116 and SW620 colorectal cancer cells by inhibiting Bcl2/caspase-9/caspase-3 pathway [34].

G protein coupled P2Y purinergic receptors is considered to be one of the cellular and molecular events that coordinate the characteristics of cancer cells. P2Y purinergic receptor activation also mediates the progress of intracellular signal transduction and regulation of CRC. P2Y1, P2Y2, and P2Y4 receptors, coupled to heterotrimer Gq protein, activated GαQ subunit-dependent IP3/DAG cascade and increased intracellular calcium concentration [Ca²⁺] and activation of protein kinase C (PKC). P2Y12, P2Y13, and P2Y14 receptors are coupled to the heterotrimer Gai/o protein, which inhibits Gαi/o-dependent adenyl cyclase activity, thereby reducing intracellular cAMP level [48]. The activation of protein GQ mediated by P2Y purinergic receptors leads to the stimulation of PLC, resulting in inositol-(1mem4pyr5)-triphosphate and diacylglycerol (DAG). Inositol-(1min4min5)-triphosphate increased intracellular calcium level. DAG stimulated PKC and possibly inhibited adenosine cyclase [49]. P2Y purinergic receptors can regulate the progression of CRC by mediating protein coupling and calcium signal. Short-term stimulation of P2Y2 receptor can induce calcium mobilization and subsequent transmembrane calcium influx in Colo320 or HT-29 colorectal cancer cells [50]. It was found that PKC activation did not contribute to receptor-mediated increase of cAMP, while PKC inhibitor stauroporine could not completely reduce the increase of cAMP induced by P2Y2 receptor [50]. Inhibition of P2Y2 receptor completely eliminated the increase of intracellular calcium in colorectal cancer cells (HT29, LS513, LS174T, and HCT116) after ATP exposure [39]. Other studies have shown that P2Y6 receptor is directly coupled to heterotrimer G13 protein and activates Gα13-dependent Rho signal transduction [51]. Another P2Y subtype, P2Y11 receptor, is uniquely coupled to Gq and Gs proteins, increasing Gαq-dependent [Ca²⁺] and Gαs-dependent cAMP levels, respectively [51].

P2X receptors regulate inflammatory response and colitis-associated cancer

Inflammatory bowel disease is a common inflammatory disease of gastrointestinal tract, and colitis associated cancer is a complication of inflammatory bowel disease [52]. Different studies have shown that P2X purinergic signals play an important role in the pathogenesis of inflammatory bowel disease. In P2X subtypes, P2X7 receptor plays a key regulatory role in inflammatory response because of its unique biological characteristics [53]. The P2X7 receptor is a key event in the control of intestinal inflammation. There is overexpression of P2X7 receptor in intestinal mucosa of patients with inflammatory bowel disease, and inhibition of P2X7 receptor has a protective effect on colitis [54]. P2X7 receptor gene knockout can induce the accumulation of regulatory T cells in the colon, and the activation of P2X7 receptor triggers regulatory T cell death [54]. Blocking Panx1 and P2X7 receptor can reduce intestinal mucosal crypt injury, tight junction loss and increase cell permeability induced by inflammatory cytokines [55]. P2X7 receptor blockade can limit the increase of pro-inflammatory mast cells and cytokines and promote the survival of regulatory T cells (Tregs) and intestinal neurons [56]. P2X7 receptor blockade changes the infiltration of proinflammatory mast cells, promotes the accumulation of Tregs in digestive system lesions and the proliferation of intestinal epithelial cells, and protects intestinal epithelial cells from apoptosis [56]. The number of mast cells expressing P2X7 receptor increased in the colon of colitis mice and Crohn’s disease patients. Treatment with P2X7 receptor specific antibodies can inhibit mast cell activation and subsequent intestinal inflammation [57].

In addition to P2X7 receptor, other P2X subtypes, such as P2X1 and P2X4, were also found to mediate colonic inflammatory response [58, 59]. One study reported that the expression of P2X1 receptor was significantly up-regulated in inflamed colon tissue. P2X1 receptor gene knockout inhibited the inflammatory response of dextran sulfate-induced colitis in mice and decreased neutrophil infiltration and intestinal microflora [58]. It was further shown that specific P2X1 receptor inhibitors successfully improved the efficacy of anti-tumor necrosis factor-α in the treatment of colitis in mice [58].

NLRP3 inflammasome and its downstream signaling factors play an important role in the progression of inflammatory bowel disease. P2X7 receptor can induce colonic inflammatory response and colitis associated colorectal cancer by regulating NLRP3 signal [60]. Therefore, preventing the overactivation of NLRP3 inflammasome may be a therapeutic approach for colitis associated colorectal cancer. BBG (P2X7 receptor selective antagonist) or OLT1177 alone can decrease the recruitment of NLRP3 inflammasome and the subsequent activation of caspase-1, IL-1β, and IL-18 and reduce ulcerative colitis in rats [61]. Herb-partitioned moxibustion can Inhibit the expression of P2X7 receptor, Pannin-1, and NF-κBp65 in colon tissue, block the abnormal activation of NLRP3 inflammasome, reduce the release of downstream inflammatory cytokines IL-1β, and ultimately inhibit colonic inflammation [62]. It was found that P2X7 receptor (+ / +) mice showed the increased accumulation of immune cells (CD4- and CD11b-positive cells), production of proinflammatory cytokines, upregulation of NLRP3 and NLRP12 genes, more ulcers, and larger tumors [10]. The possible mechanism of up-regulation of P2X7 receptor involved in the progression of colitis associated colorectal cancer combines with the signals of intestinal microflora, which leads to the activation of inflammasome, and amplifies the inflammatory response. Activation of P2X7 receptor-NLRP3 signal and infiltration of circulating leukocytes and mononuclear macrophages increased the expression of transforming growth factor-β and fibroblast growth factor-1, resulting in peritoneal spread of colon cancer and ovarian cancer [63].

P2X purinergic receptors and the progression of CRC including growth and apoptosis

Activation of P2X purinergic receptors promotes the growth, invasion, and metastasis of CRC. Among P2X subtypes, P2X7 receptor is still favored by many researchers and has received extensive attention in the study of the mechanism of growth and metastasis of CRC. P2X7 receptor activation increases the expression of Vimentin, Snail, and Fibronectin, reduces the expression of E-cadherin, and promotes the metastasis and EMT formation of colorectal cancer [35]. Our previous studies have found that ATP or BzATP activates P2X7 receptor and promotes the growth and metastasis of LoVo and SW480 colon cancer cells [47]. Tregs metastasis or transient depletion of NK cells can significantly increase the expression of IL-22 and inflammatory signal molecules P2X7 receptor in colorectal tumors [64]. P2X7 receptor antagonist A438079 decreased the expression of apoptosis-related markers (Bcl2, BAX, caspase-9, cleavage caspase-9, caspase-3, and cleavage caspase-3) and pyroptosis-related markers (NLRP3, Asc, caspase-1, and IL-Iβ) of HCT-116 and SW620 cells, thus inhibiting the proliferation, invasion, and migration of colorectal cancer [34]. Other studies have shown that overexpression of P2X7 receptor promotes the induction of NF-κB-dependent cytokines, which leads to the recruitment of tumor-associated macrophages and promotes the growth, progression, and stromal remodeling of colorectal cancer [65] (Fig. 1).

Fig. 1.

P2X purinergic receptors (P2X7 receptor) activation regulating the progression of CRC. Tumor microenvironment is rich in ATP, which activates P2X7 receptor. P2X7 receptor activation can lead to the opening of ion channels (mainly calcium influx), which promotes the growth of CRC by mediating calcium mobilization. P2X7 receptor activation can promote the invasion and metastasis of CRC by activating different intracellular signal pathways (such as P13/AKT, STAT3, and Bcl2/caspase) and regulating the expression of metastasis/EMT-related genes (such as Vimentin, Snail, and Fibronectin). However, under the stimulation of continuous activator, the ion channels of P2X7 receptor gradually open into larger membrane pores, which can lead to structural changes and the imbalance of intracellular molecular metabolism, and eventually lead to CRC cell apoptosis. Therefore, P2X7 receptor plays a promoting or inhibitory role in the regulation of CRC

Angiogenesis provides the nutritional and environmental basis for tumor growth and promotes tumor growth and metastasis. The expression of VEGF and EGFR is up-regulated in colorectal cancer, and their expression level is related to liver metastasis of colorectal cancer [66], while blocking VEGF or EGFR can inhibit proliferation and metastasis of colorectal cancer [66]. The mouse model of xenotransplantation in vivo showed that the combination of anti-VEGF antibody and anti-EGFR antibody could significantly inhibit the formation of colorectal cancer and angiogenesis [66]. Overexpression of P2X7 receptor can increase the expression of CD31 and VEGF and promote angiogenesis in colorectal cancer [65].

Interestingly, other studies have revealed the opposite results. Activation or overexpression of P2X7 receptor promotes apoptosis of colorectal cancer. Indeed, the activation or up-regulation of P2X7 receptor can also inhibit the growth of colorectal cancer. Hyperthermia (40 ℃) can enhance the eATP-mediated killing effect on MCA38 colon cancer cells. This may be related to the increase of membrane fluidity induced by high temperature, which enhanced the function of P2X7 receptor and the opening of pores [67]. The tumor growth and metastasis and diffusion rate of P2X7 receptor gene deficient mice were significantly accelerated, the release of IL-1β and VEGF was significantly decreased, and the inflammatory cell infiltration almost disappeared completely [68]. After short-term hypotonic stress, ATP released by human colon cancer cells (SW403, HCT-116, and Colo) specifically binds to P2 purinergic receptors (possibly P2X7 receptor), resulting in apoptosis (induced by transient cell swelling) [69]. This apoptosis is mediated by the activation of caspase-3, caspase-8, annexin V, and the release of cytochrome c induced by extracellular ATP.

Other P2X purinergic receptors (P2X3, P2X4, and P2X5 receptors) may play a regulatory role in the growth and metastasis of colorectal cancer [31, 39, 70]. High dose of organic nitroglycerin trinitrate (GTN) is the donor of nitric oxide (NO), which can induce apoptosis of human colorectal cancer cells (SW480, CT26, and C51). The inhibition of P2 purinergic receptors by suramin and its competition with ATP/UDP attenuate this toxic effect. P2X3, P2Y1, and P2Y6 receptors may play a certain role in this cytotoxic effect [31]. The death of colorectal cancer cells (CT26 and HCT-116) induced by chemotherapy leads to the release of ATP, which triggers the P2X4 receptor-mediated mTOR-dependent survival process in adjacent cancer cells [71].

Therapeutic effect of antagonizing P2X purinergic receptors on CRC

Activation of P2X7 receptor can regulate tumor progression. Therefore, using its antagonists (such as A438079, AZD9056, A740003, and AZ10606120) can antagonize its activity and play an inhibitory pharmacological role in most tumors (prostate cancer, lung cancer, and pancreatic cancer) [72–74]. Similarly, these partial antagonists have also been used in the treatment of CRC. A438079, a selective antagonist of P2X7 receptor, 10 μm can significantly antagonize the activity of P2X7 receptor and inhibit the proliferation, migration, and invasion of colon cancer cells (SW620 and HCT-116) [34]. We previously studied that the use of P2X7 receptor antagonists (A438079 and AZD9056) 10 μm or siRNA to knock down the expression of P2X7 receptor inhibited the proliferation, migration, invasion, metastasis, and EMT formation of colon cancer cells (LoVo, SW620, SW480, and HCT-116) [35, 47]. Intraperitoneal injection of P2X7 receptor antagonist A74003 (10 μmol/L) or AZ10606120 (300 nmol/L) has a strong pharmacological effect on inhibiting the growth of colorectal cancer [68]. It also shows that P2X7 receptor antagonists still have strong anticancer activity even after systemic administration. P2X7 receptor (+ / +) mice treated with P2X7 receptor antagonist A740003 exhibited the increased CD4- and CD11b-positive immune cell accumulation and produced less inflammation, which induced more ulcers and colitis-associated colon cancer formation [10]. Another study shows that emodin is a natural anthraquinone, which can reduce the activation of M2-like tumor macrophages by antagonizing P2X7 receptors in bone tissue, inhibit cell recruitment to tumor microenvironment, and prevent the development of colorectal cancer [75]. These studies suggest that P2X purinergic receptor (P2X7 receptor) antagonists may have pharmacological effects on CRC, but the exact mechanism by which these antagonists affect and alter the behavior of CRC cells is unclear. Nevertheless, the development of strong P2X7 receptor antagonists may become a potential pharmacological target for CRC therapy.

Functional role of P2Y purinergic receptors in CRC

The role of P2Y purinergic receptors in tumor progression, including growth, migration, invasion, and metastasis, has been confirmed by different studies. Long-term application of P2Y2 receptor agonists can induce an increase in the apoptosis rate of Colo320 or HT-29 colorectal cancer cells in a time-dependent manner, resulting in the inhibition of cell proliferation of Colo320 or HT-29 cells [50]. P2Y2 receptor resists apoptosis induced by ursolic acid in colon cancer cell HT-29. Ursolic acid induces the increase of intracellular ATP and P2Y2 receptor transcription, activates Src and p38 phosphorylation, leads to overexpression of COX-2, and induces resistance to apoptosis in HT-29 [76]. P2Y6 receptor murine mice showed a decrease in the number of colorectal cancer and a significantly smaller tumor volume. P2Y6 receptor stimulated by MRS2693, a selective agonist, can protect HT-29 cells from apoptosis induced by TNF-α [77]. This protective effect is achieved through the stable phosphorylation of X-linked inhibitor of apoptosis protein (XIAP).

An important factor in the growth and metastasis of CRC is tumor angiogenesis. Platelets are an often neglected component of the immune system and have been shown to promote tumor growth [78]. Platelets infiltrate into the tumor microenvironment and interact with tumor cells in a cadherin-6-dependent manner, resulting in platelet diffusion, release of its particle contents, and production of three kinds of particles expressing platelet markers and/or tumor markers (IMP). The presence of iMP was confirmed in colorectal cancer tissue specimens. [79]. In blood, IMP activates endothelial cells and platelets, induces the transformation of swollen HT-29 colorectal cancer cells from epithelium to mesenchymal cells, and promotes metastasis [79]. The interaction between platelet GPVI and Galectin-3 expressed by tumor cells makes use of the tyrosine activating motif of immune receptor in platelets, which is beneficial to the metastasis of MC38 colorectal cancer cells and breast cancer cells (AT3 and E0771) [80]. P2Y12 receptor, an important member of P2Y subtype, is enriched and expressed in platelets, which plays a key role in maintaining platelet function in hemostasis and thrombosis. Different studies have proved that P2Y12 receptor is expressed in a variety of tumor cells and regulates tumor progression by mediating the interaction between platelets and tumors [81–83]. Caco-2 colorectal cancer cells can induce dense granules to secrete ADP. Stimulation of P2Y12 receptor is considered necessary for complete platelet aggregation [84]. A small amount of thrombin in plasma drives platelet activation and secretion, which is mediated by the positive feedback signal secreting ADP [84]. The anti-hepatoma effect of platelets is mediated by CD40L release dependent on P2Y12 receptor, which leads to CD40 receptor activating CD8 + T cells. Similarly, P2Y12 receptor is expressed in colorectal cancer and plays a certain role in tumor angiogenesis by regulating platelet function. The application of P2Y12 receptor antagonist (Aspirin) can reduce the proliferation ability of HCT-116 colorectal cancer cells [85]. HT-29 colorectal cancer cells induced cell masses dominated by platelet-platelet aggregates, while ticagrel (P2Y12 receptor antagonist) significantly inhibited platelet-platelet aggregation induced by tumor cells [86]. It is suggested that the inhibitory effect of P2Y12 receptor may reduce the spontaneous platelet aggregation and activation in patients with metastatic cancer. The inhibitory effect of Aspirin or ticagrel on platelet COX-1 in mice can prevent the increase of metastasis rate caused by platelet induced TXA2 and PGE2 production in HT-29 colorectal cancer cells in vitro [87] (Fig. 2). These studies reveal a fact that P2Y purinergic receptors regulate the progression of CRC. Therefore, targeting P2Y purinergic receptors may be another molecular target for the treatment of CRC.

Fig. 2.

Activation of P2Y purinergic receptors (P2Y12 receptor) promotes the growth and metastasis of CRC. Tumor microenvironment is rich in ATP. These ATP can be produced by tumor cells, macrophages, T lymphocytes, and fibroblasts and cleaved into corresponding products (AMP, ADP, and Ado) by the action of CD39 and CD73. ADP is a powerful activator of P2Y12 receptor. P2Y12 receptor activation regulates the level of intracellular cAMP and produces platelet secreting factor (COX-1) by mediating calcium signal. P2Y12 receptor activation promotes platelet activation, including migration, VEGF, and other secreting factors (TAX2), promotes the growth of CRC blood vessels, and provides a good basic environment for CRC growth and metastasis. In addition, tumor cells or activated platelets can further produce ATP, and these ATP can further cleave into ADP to activate P2Y12 receptor, forming a positive feedback loop and promoting the progress of CRC, while the application of P2Y12 receptor antagonists (Aspirin and ticagrel) can antagonize the activity of P2Y12 receptor, which can partially reverse the above corresponding phenomena, inhibit the growth and metastasis of CRC, and produce certain therapeutic pharmacological effects

Conclusion

The functional role of P2 purinergic receptors in CRC has been confirmed by different studies. ATP and its cleavage products in tumor microenvironment can be used as activators of P2 purinergic receptors to regulate the behavior of tumor cells and non-tumor cells. The role of P2 purinergic receptors in regulating the function of CRC includes activating different signal transduction, immunomodulation, and promoting angiogenesis. The application of P2 purinergic receptors antagonists can inhibit the growth or metastasis of CRC. In addition, the high expression of P2 purinergic receptors (P2X7 receptor) in patients with CRC is closely related to the clinicopathological features and can be used as a molecular marker for predicting CRC. Importantly, P2X7 receptor plays a dual role in CRC, that is, promotion or inhibition. But the specific mechanism is not clear, which may be related to the molecular structure of P2X7 receptor. In its low activity state, open ion channels activate intracellular signal transduction and promote tumor progression, while under the continuous stimulation of activators, its activity and molecular configuration change, and ion channels gradually increase, resulting in the imbalance of cell membrane stability and fluidity, leading to cell apoptosis. Another possible correlation is the activity of immune cells mediated by P2X7 receptor, which leads to immune escape or immunosuppression of CRC. Therefore, these factors should be taken into account in the application of P2X7 receptor in the treatment of CRC. Moreover, P2Y12 receptor mediates platelet activity in the progression of CRC. In short, P2 purinergic receptors play an important role in CRC and may be used as a potential molecular target for the prevention and treatment of CRC.

We n-jun Zhang

Doctorate degree, engaged in the study of gastrointestinal tumors for many years, has a good theoretical basis in the field of pathogenesis and progression of gastrointestinal tumors.

Author contribution

Wen-jun Zhang: Completed the writing and the final manuscript, reviewed and revised the article.

Li-peng Zhang: Completed the data collection and drafted the paper.

Si-jian Lin: Completed the review and revision.

Cheng-yi Wang: Complete the references and revision.

Yi-guan Le: Completed the revision and guidance.

Funding

These studies were supported by grants from the Youth Science Foundation of Jiangxi Province (20224BAB216030), the Project of Education Department of Jiangxi Province (GJJ2200238), the Natural Science Foundation of Jiangxi Province (20232BAB206048), and the Jiangxi Province Traditional Chinese Medicine Science and Technology Plan (2023B1213).

Data Availability

All data generated or analyzed during this study are included in this article. And we have not used other data that has already been published. All the data presented in this article are original results derived from this study.

Availability of supporting data

All data generated or analyzed during this study are included in this article, and we have not used other data that has already been published. All the data presented in this article are original results derived from this study.

Compliance with ethical standards

Competing interests

The authors declare no competing interests.

Conflict of interest

Wen-jun Zhang declares that he/she has no conflict of interest.

Li-peng Zhang declares that he/she has no conflict of interest.

Si-jian Lin declares that he/she has no conflict of interest.

Cheng-yi Wang declares that he/she has no conflict of interest.

Yi-guan Le declares that he/she has no conflict of interest.

Ethical approval and consent to participate

Ethical approval has been exempted by the Ethics Committee of the Second Affiliate Hospital of Nanchang University. All protocols were approved by the Animal Care and Ethics Committee, China.

Consent for publication

Not applicable.