DNA structures extruded from endothelial cells were observed during arterio-arterial embolization, a pathological condition occurring following cholesterol crystal-induced embolism in the kidney (231). Cholesterol crystal embolism is mobilized from an atherosclerotic plaque, followed by vessel obstruction, ischemia and organ failure (232). Studies by Shi et al., showed that injection of cholesterol crystals into the artery of the mouse kidney generates a thromboinflammatory environment with the presence of intravascular thrombi, composed of platelets, fibrin, neutrophils and extracellular DNA (231). Using in vitro cell culture experiments, exposure of neutrophils to cholesterol crystals or the supernatant of cholesterol crystal-activated platelets induced neutrophil necrosis and the release of chromatin and DNA to the cell culture supernatant (231). Interestingly, exposure to increasing doses of cholesterol crystals also induced necrosis of glomerular endothelial cells and consequent DNA release (231).

The vasculature of metastatic organs is frequently damaged and metastases can induce cell death (233). Necroptic cell death and subsequent DNA release occur in endothelial cells, involving RIPK1, RIPK3 and MLKL cell death signaling pathways. Tumor cell-induced endothelial necroptosis was shown as an important mediator of tumor cell extravasation and subsequent tumor metastasis (234). Further experiments need to be performed whether under certain conditions endothelial cells may also undergo ETosis.

Platelets lack nuclear DNA and the amount of mitochondrial DNA is very limited, due to the few numbers of mitochondria per platelet (235). Theoretically, accumulated platelets at the injury sites may release mitochondrial DNA upon platelet activation (236, 237). This extracellular DNA may be contributed to immune cell-derived ETs, and further amplify cancer-associated thrombosis, thromboinflammation and tumor progression. Further studies are important to establish the role of platelet-derived ETs in these processes.

Cancer is associated with cachexia, vascular and metabolic dysregulation of the heart (238, 239). Cardiomyocytes possibly are a major source of extracellular DNA in patients with myocardial infarction (240, 241). Microvesicles and exosomes released from cardiomyocytes also contain extracellular DNA (242). Due to the limited experimental evidence, further studies are necessary to investigate the role of cardiomyocyte-derived extracellular DNA, analyze metabolic and DNA contents in patients with cancer and establish the contribution of ETs in myocardial infarction and cancer-associated heart dysfunction.

The blood plasma levels of extracellular DNAs are increased in human patients with breast, melanoma, pancreatic and colon cancers, which are directly extruded by cancer cells (4, 10, 243). Circulating extracellular DNA can interact with several molecules, exposed on the surface of blood cells, leading to the penetration of DNA (244, 245). Histones and complement factors directly bind and capture DNA (246–248). DNA can also be transferred to the exosomes and microparticles and secreted to the circulation. Indeed, circulating microvesicles isolated from the blood cancer patients contain fragments of mutated genes, such as phosphatase and tensin homolog (PTEN), p53 and KRAS (249–251). Cai and colleagues found that BCR/ABL hybrid genes can be transferred from chronic myeloid leukemia cells to the HEK293 and neutrophils, increasing DNA coding mRNA and protein levels (252). Similar results were observed with vascular smooth muscle cells and leukocyte-derived extracellular vesicles delivering the angiotensin receptor type 1 (AT1R) gene DNA to HEK293 cells and sex-determining region Y (SRY) DNA into the endothelial cells (253, 254).

DNAse enzymes are divided into two major families, DNAse I and DNAse II. Although DNAse I is found in exocrine gland secretions and blood, DNAse II derives from lysosomes/phagolysosomes (255). Regarding the sources of circulating DNA, it was assumed that tumor cells in cancer patients shed and release DNA into the bloodstream and this correlated with the pathogenesis of the disease (5, 256). In line with this, DNAse I levels in cancer patients are elevated during remission, and after successful interventions and decreased during cancer progression and metastasis. Furthermore, failure of DNAse levels to increase in response to treatment was correlated with poor prognosis (257, 258). However, DNAse activity in the blood was found to differ between healthy subjects and cancer patients and also varies between cancer types and stages of cancer (257–261). Indeed, decreased DNAse activity was found in patients with malignant lymphoma, gastrointestinal and prostate cancer (260, 262, 263), while the levels of DNAse activity were higher in breast cancer patients compared to the control (264). The physiological relevance of DNAse function in NETosis was proved in knockout mouse models. Mice with DNAse I and DNAse I like-3 enzyme deficiencies developed NETosis with intravascular clots and obstructed blood vessels which resulted in tissue damages of vital organs, such as the lung, liver and kidney (265). In humans, genetic mutations of DNAse are associated with autoimmune diseases such as systemic lupus erythematosus (266). DNAse X/Apo10 antibodies were found in patients with oral squamous cell carcinoma, indicating gene inactivation of DNA X in this type of tumor (267). A therapeutic strategy based on the delivery of transgenic vectors expressing DNAses was proposed to target DNA destruction or apoptosis. In 2011, Karli Rosner suggested an anti-cancer therapeutic approach based on human recombinant DNAse I. According to his approach, the replacement of apoptosis-activated endogenous DNAses with human recombinant DNAse I might help to bypass cancer defense mechanisms, increasing the killing efficiency of chemo and radiotherapy-resistant tumor cells (268). Since the inactivation of endogenous DNAse X gene was found in many tumor cells types, the strategies to restore the levels of DNAse X in cancer cells could be an important targeted therapy (267, 269). Delivery of vectors encoding several DNAses under one common promoter into the cancer cells could successfully induce apoptosis (268, 269).

Based on these findings, gene therapy was developed in a mouse model of colorectal cancer in which an adeno-associated virus (AAV) vector was used to express DNAse I in the liver, thereby suppressing the development of hepatic metastases. After AAV-DNAse I treatment, NETosis was inhibited in the tumor tissues with restored local immune responses by increasing the percentage of CD8+ T cells (270).

Recombinant DNAse I has been successfully used as an anti-cancer agent and studied as a prognostic/diagnostic marker during cancer therapy. In 1961, de Lamirande determined the effect of DNAse and RNAse in mice bearing Ehrlich ascites carcinoma for the first time (271). After tumor cell implantation, daily injection of DNAse I could increase the survival rate of treated mice, but RNAse treatment did not affect mouse survival. A hypothesis was proposed, which included the uptake of DNAse into cancer cells, followed by necrosis and digestion of nuclear DNA (271). In other studies, the daily injection of RNAse and DNAse alone or in combination could enhance nuclease activity of blood plasma of tumor-bearing mice, and decrease the levels of extracellular DNA, back to the levels of control animals. Degradation of DNAs in the blood plasma was associated with reduced metastasis of LLC and hepatoma A–1 (HA-1) cancer cells (272–274). In the model of LLC, exogenous DNAse treatment not only inhibited metastasis but also increased DNAse activity in the blood, destroying extracellular DNA in the circulation of tumor-bearing mice by targeting tumor-associated DNA fragments such as short and long interspersed retrotransposable elements (SINEs and LINEs) and also oncogenic sequences (274, 275). Furthermore, daily intramuscular injection of bovine pancreatic DNAse I in LLC tumor model could also strongly decrease metastasis (276). In mouse models of melanoma, lymphosarcoma or pancreatic cancer, DNAse I treatment had also strong anti-tumor and anti-metastatic effects by destroying extracellular DNA (275, 277, 278). Bovine pancreatic DNAse also displayed anti-metastatic effects inhibiting the number of lymph nodes and lung metastasis in mouse models of leukemia and lymphoma cancers. Although bovine pancreatic DNAse I could inhibit the proliferation of several cancer cell types (Calu-1, SK-MES-1, HeLa, HEp-2 and L-929), it did not affect the peripheral blood mononuclear cells and fibroblasts (279). Combined treatment of DNAse I with proteases such as papain, trypsin or chymotrypsin led to a significant decrease of DNA content in the blood serum of rats, and no anti-tumor effects were observed in mice treated with proteases alone (280).

Pancreatic cancers belong to the group of diseases which affect both the endocrine and exocrine functions of the pancreas (281). The tumor microenvironment is instrumental in pancreatic tumor growth and metastasis. Although some mechanisms reflect tumor cell-autonomous processes, most require the interaction of tumor cells with tumor microenvironment, including endothelial cells, fibroblasts, and immune cells (282). In addition, chronic inflammation, thromboembolism and hypercoagulability are known as key features of PDAC (283, 284). Interestingly, DNAse I treatment of pancreatic cancer cells could strongly decrease tumor cell adhesion and migration, although tumor cell proliferation was not affected. In the orthotopic pancreatic cancer model, DNAse I treatment also strongly inhibited tumor burden and tumor metastasis to the liver and diaphragm, confirming the important pathological role of extracellular DNA in pancreatic cancer. Elevated CXCL8 secretion was detected in the medium of pancreatic cancer cell lines derived from liver metastases, in comparison with immortalized pancreatic ductal epithelial cells. Furthermore, the treatment of pancreatic cancer cells with recombinant CXCL8 could strongly increase extracellular DNA production (285). CXCL8 also induces ET formation in neutrophils, thereby enhancing cancer malignancy (14, 44, 96). DNAse I treatment strongly reduces ETs, and also the percentage of polymorphonuclear neutrophils that released observable ETs (286). Pancreatic tumor-bearing mice had also increased levels of NETs, and more rapid thrombotic occlusion in the injury model of jugular vein. DNAse I did not affect thrombotic occlusion in control mice, but protected tumor-bearing mice from enhanced venous thrombosis (287). These results suggest that enhanced NETosis contributes to thrombosis in pancreatic cancer.

Interestingly, DNAse I can also inhibit thrombosis independently of neutrophils. In the mouse model of cholesterol crystal embolism, in vivo depletion of circulating neutrophils in the peripheral blood did not influence the severity of disease, but DNAse I treatment significantly inhibited the numbers of obstructed vessels, decreased ischemic organ failure and kidney infarction. Preincubation of washed platelets with DNAse I inhibited platelet activation, P-selectin exposure, aggregation response to collagen, collagen-related peptide or thrombin. In addition, DNAse I-treated platelets formed less fibrin. DNAse I treatment also reduces the levels of secreted adenosine triphosphate (ATP) in human and mouse platelets, which strongly inhibits platelet aggregation, and ATP-dependent neutrophil activation (231, 288). Earlier, it was proposed that neutrophils are required for thrombosis in the laser-induced arterial injury model (289). Although DNAse I treatment induced the hydrolysis of ATP and adenosine diphosphate (ADP), decreasing fibrin formation and inhibiting thrombosis, scanning electron microscopy did not reveal classical NET structure in this thrombosis model (288).

Polyphosphate (polyP) is synthesized enzymatically from ATP and this metabolic conversion is fully reversible. PolyP is stored in dense granules of platelets, and secreted upon platelet activation. Extracellular polyP accelerates the coagulation cascade by factor V activation, promotes factor XI activation through thrombin and blocks the anti-coagulant activity of tissue factor inhibitor (TFI), thereby enhancing blood clotting (290). Interestingly, DNAse I could decrease ATP and polyP levels in vitro (231, 288, 291), indicating that DNAse I may inhibit ATP metabolism, or enhance ATP degradation or conversion of ATP to adenosine monophosphate (AMP). Altogether, these results suggest that the anti-thrombotic effects of DNAse I treatment in platelets may occur in an ATP/polyP-dependent manner.

Several experimental studies using cancer and thrombosis mouse models suggested that targeting extracellular DNA with DNAse I may offer a potential anti-cancer and anti-thrombotic strategy (278, 280, 292). However, only limited clinical studies with DNAse I treatment have been reported so far. In patients with cystic fibrosis, nebulized recombinant human DNAse treatment could reduce sputum viscosity and improve pulmonary function (293, 294). Therefore, recombinant human DNAse treatment is recommended in patients with cystic fibrosis and also in patients with other moderate or severe suppurative lung diseases. Further investigation is necessary whether DNAse I treatment may be effective in cancer or cancer-associated thromboinflammation.

Aberrant arachidonic acid metabolism is involved in the inflammatory and carcinogenic processes (295). Aspirin (acetylsalicylic acid) irreversibly acetylates and thus inhibits the enzymatic activity COXs, thereby blocking the conversion of arachidonic acid to thromboxane A2 (TxA2) (296). In mouse models, aspirin treatment prevents NET-induced injury of the lung endothelium by inhibiting platelet activation and NETosis (297). A higher bacteria count in the blood was detected in aspirin-treated mice after infection, indicating that aspirin may interfere with NET functionality. However, this action of aspirin may be independent of platelet-resident COX activity, since aspirin-treated neutrophils had impaired NETosis (297, 298).

Prostaglandin E2 (PGE2) is a prostanoid fatty acid metabolic product of arachidonic acid. PGE2 inhibits PMA-induced NETosis through prostanoid receptors of EP2 and EP4 (299). Studies by Domingo-Gonzalez et al., showed that murine bone marrow transplant neutrophils which overexpress COX2 induce defective bacteria clearance (300). When these neutrophils were stimulated with PMA or rapamycin, NETosis was strongly reduced compared to control. After bone marrow transfer, NET formation was rescued using COX inhibitors. The same effect was achieved via EP2 receptor antagonist (PF-04418948) or EP4 antagonist (AE3-208) in neutrophils from bone marrow transplant mice and hematopoietic stem cell transplant patients (300).

In mice and healthy donors, NETosis was also inhibited by exogenously injected PGE2 which was dependent on the cAMP-PKA pathway (299, 300). Consistently, incubation of neutrophils with cAMP analog dibutyryl-cAMP, rolipram or butaprost could also inhibit NETosis (299).

Chloroquine and hydroxychloroquine are anti-malarial drugs, which appeared as promising treatments also for cancer (301). Chloroquine inhibits autophagy in different cell types including neutrophils (127). Several groups have shown that autophagy promotes NETosis (62, 118, 302–304). However, studies using pharmacological inhibitors of autophagosome acidification and neutrophil- and eosinophil-autophagy‐related 5 (ATG5) conditional knock-out mice could not confirm these results (146). Chloroquine treatment reduces the severity of acute pancreatitis in mice, thereby improving survival (305). In cell culture assays, chloroquine could not diminish NETosis, indicating an indirect mechanism (306). Hydroxychloroquine is also known as an anti-inflammatory drug, which can block TLR/COX2 pathway-dependent NET formation and consequent metastasis in hepatocellular carcinoma (2, 107, 307). In the mouse model of PDAC, chloroquine treatment reversed hypercoagulability by reducing NET-mediated platelet aggregation and the release of circulating TF. Patients treated with hydroxychloroquine on a randomized protocol of preoperative chemotherapy showed a reduction in pre-operative VTE rate (127). Although several clinical trials showed the benefits of chloroquine as an anti-tumor drug (308), the precise molecular mechanisms of chloroquine-mediated effects has not been established. It was proposed that chloroquine may influence autophagy (309). Chloroquine in combination with other chemotherapeutic drugs could increase the efficiency of drug treatment, although it can accelerate chemotherapy-associated organ injury (301). Therefore, it is important to further investigate the effects of chloroquine on cancer-induced NETosis, thromboinflammation and organ injury.

Bacterial infection of host tissues activates neutrophils and induces NET formation, thereby activating the innate immune system, including macrophage phagocytosis. Interestingly, Staphylococcus aureus can escape from NETs, thus converting NETs to deoxyadenosine, thereby inducing immune cell death by caspase-3-mediated mechanism. Staphylococcus aureus can secret nuclease and adenosine synthase which modifies the structure of NETs, thereby destroying the NET-mediated immune defense system (310). Staphylococcus aureus also produces a plasminogen activator staphylokinase, which is a fibrin-specific thrombolytic biomolecule (311). Staphylokinase was proposed for the therapy of stroke and myocardial infarction. However, it has a short life-time in the blood, which limits the clinical application. Strategies based on the PEGylation (attachment of polyethylene glycol) may prolong the half-life time of staphylokinase, thereby improving its bioactivity in disease conditions (312).

Cl-amidine and F-amidine target all peptidyl arginine deiminase (PAD) isoforms were actively applied in many preclinical models to study NETosis. Various tumors are associated with the overexpression of PAD and increased citrullination. In 1958, Rogers and Simmonds were the first to describe protein citrullination in an animal protein as the process of converting peptidyl arginine into peptidyl citrulline. Since citrulline cannot be encoded in vivo, it only occurs after translation (313). Peptidyl arginine deiminases (PADs, also called PADIs) are an enzyme family which can convert protein arginine residues to citrulline in a Ca²⁺-dependent manner. This enzyme family comprises 5 isoforms (including PAD1, 2, 3, 4 and 6) which are highly conserved, have tissue-specific distribution and target substrates respectively (314).

PAD2-mediated histone citrullination is proposed as a potential therapeutic target for prostate and colon cancer (201, 315). PAD2 also regulates genes expression related to lactation through histone citrullination (316). The gonadotropin-releasing hormone (GnRH) agonist can stimulate PAD2-mediated histone H3 citrullination which epigenetically regulates the expression of gonadotropin genes such as luteinizing hormone β (LHβ) and follicle-stimulating hormone β (FSHβ) in gonadotropes (317). Recent studies have identified that PAD2 inhibition can reduce inflammatory cytokine production and NET formation in endotoxemia (318). PAD4-mediated citrullination promotes chromatin decondensation and DNA fragmentation, thereby affecting chromatin structure. PAD4 is critical for NET-mediated anti-microbial function (319). Furthermore, PAD4 can also regulate the transcriptional activity of p53 in tumor progression (320). Additionally, PAD4 promotes the metastasis of gastric tumors by regulating the expression of CXCR2, keratin K14 (KRT14) and TNFβ, which can accelerate angiogenesis, cell proliferation, migration and tumor immune microenvironment establishment (321). Inhibition of PAD4-mediated NETosis was also possible using an antagonist miR-155, which inhibits PAD4 mRNA synthesis and NET formation in response to PMA (322). In the experimental model of systemic lupus erythematosus, Cl-amidine treatment strongly inhibits NET-induced vascular damages, endothelial dysfunction and kidney injury. Inhibition of PAD4 also strongly decreased the expression of IFNγ, reduced proteinuria and immune complex attachment to the kidney tissues and in addition, protected from skin disease (323). Interestingly, PAD4-deficient mice had accelerated diabetic wound healing compared to wild-type mice (324). Although these irreversible inhibitors inactivate Ca²⁺-bound PAD4, they lack specificity and also interact with other isoforms of the PAD-family. Lewis et al., generated two reversible inhibitors GSK199 and GSK484 which are highly specific for PAD4 and can inhibit NETosis in murine and human neutrophils (325). Removal of NETs with DNAse I or pharmacological inhibition of PAD4 with GSK484 inhibitor prevent cancer-associated kidney injury in mice (326). However, in recent studies, GSK484 also enhanced irradiation-induced damages in triple-negative breast cancer cells, which subsequently had inhibitory effects on cell proliferation, migration and invasion (327). In mouse models of sepsis, deficiency of PAD4 or DNAse I treatment strongly reduced intravascular thrombin activity, inhibited platelet aggregation and improved microvascular perfusion (328). Patients with acute thrombotic microangiopathies displayed low plasma levels of DNAse I compared to the healthy subjects (329). In mouse models of HIT, genetic deficiency or GSK484-mediated inhibition of PAD4 abolishes thrombus formation (135). In vitro, DNAse I/GSK484 strongly inhibited the epithelial-mesenchymal transition-promoting ability of NETs in gastric cell cultures (87), indicating multiple effects of exogenous DNAse I in cancer.

Other effects may also result from PAD-mediated inhibition of ET formation. PAD1 and PAD3 target keratin K1, filaggrin and myelin, thus playing a specific role in epidermis differentiation (330). PAD enzymes are also positively associated with diffuse inflammation in the brain (331). In macrophages, PAD2 becomes activated due to increased levels of Ca²⁺ and can induce apoptosis by citrullinating vimentin (332). PAD2 citrullinates many proteins such as actin and vimentin in dendritic cells and dendritic cell-derived osteoclasts and in brain tissues (333, 334). Furthermore, overexpression of PAD2 in T cell line was shown to induce vimentin citrullination and apoptosis (335). Recently, PAD3 was found to be necessary for apoptosis-inducing factor (AIF)-mediated apoptosis in human neural stem cells (336). In comparison to other PAD family members, PAD4 has more catalytic substrates. PAD4 is involved in cell apoptosis and differentiation and deiminates nonhistone proteins such as p300, nucleophosmin (NPM1), an inhibitor of growth protein 4 (ING4) and Lamin C, which are involved in cell apoptosis or DNA damage (337). Moreover, PAD4-mediated citrullination participates in the regulation of human 40S ribosomal protein S2 (RPS2) and ribosome assembly (338). PAD4 targets collagen and decreases the adhesion of synovial fibroblasts and mesenchymal stem cells (339). DNA methyltransferase DNMT3A can be citrullinated by PAD4, which provides a novel mechanism for controlling de novo DNA methylation (340).

Cyclosporine A suppresses immunocompetent T cells reversibly and is applied for the treatment of autoimmune diseases such as rheumatoid arthritis, and further viral, fungal and parasitical infections (341). Cyclosporine A binds to cyclophilin, thereby downregulating the nuclear factor of activated T cells (NFAT) signaling, thus further inhibiting the calcineurin pathway (342). Efficient induction of NETosis requires cytoplasmic Ca²⁺ increase, linking the cyclosporine A-induced calcineurin pathway to NETosis. IL8-induced NETosis is reduced by combining treatment of ascomycin and cyclosporine A (343), suggesting a possibility to develop a therapeutic approach of NETosis.

Heparin is an anti-coagulant, extensively used in different therapies for the prevention of blood clotting during heart surgery, kidney dialysis, as well as for the treatment of VTE, heart attacks and angina (344). Heparin also inhibits many hallmarks of cancer, such as cancer cell survival, angiogenesis and migration (345). Moreover, heparin treatment can induce HIT, which is a life-threatening process, based on a severe immune reaction to heparin, characterized by thrombocytopenia and severe thrombosis. In patients with HIT, antibodies are produced against heparin-platelet factor 4 (PF4) complexes. Interestingly, this immune complex can directly activate neutrophils and enhance NET formation, which is sufficient for the development of thrombosis (135). PF4 binds to NETs, which renders NETs more compact. The complex then binds HIT antibodies, thereby protecting NETs from DNAse degradation (346). In the mouse model of HIT, inhibition of NET formation through PAD4 inactivation can reduce venous thrombus formation but not thrombocytopenia (346), suggesting that other alternative molecular mechanisms are involved in this process. HIT-induced NETosis is further enhanced by ROS production and NE. Interestingly, heparin derivatives, such as low molecular weight heparin, fondaparinux and heparan sulfate cannot induce profound NETosis (347). Altogether these results suggest that heparin induces neutrophil activation and NETosis contributes to venous thrombosis in HIT, which is triggered by PF4-NET-HIT antibody complexes.

The effects of unfractionated heparin, low-molecular-weight heparin (LMWH), e.g., parnaparin and non-anti-coagulant heparin were studied in histone-induced diseases. Heparin was able to protect mice and rats from organ and tissue damage, as well as death by antagonizing histones in the blood (348–351). In a mouse model of sepsis, heparin pretreatment could significantly decrease the level of NETs in serum and lung tissues (352). NET formation promotes cancer cell migration, invasion and angiogenesis, which were inhibited by heparin or other histone-binding agents (16). NETs also contributed to a variety of cancer or cancer-associated thrombosis (97). LMWH is currently the preferred treatment for prophylaxis and cancer-associated thrombosis (353). However, more experimental evidence is necessary to understand the effects of heparin in NET formation of cancer patients. Heparin derivatives may be a promising tool to cure diseases with high levels of plasma histones, thereby potentially inhibiting NETosis without dramatic changes in hemostasis.

Metformin was originally used in diabetic patients to normalize blood glucose levels. The anti-diabetic effects of metformin are due to the inhibition of hepatic gluconeogenesis, which is possibly associated with an insulin-mediated increase in glucose uptake in skeletal muscle cells (354). Metformin acts by inducing adenosine monophosphate-activated kinase (AMPK), an enzyme regulating energy metabolism through activation of glucose or oxidation of fatty acids (355). High glucose and hyperglycemia increase the release of NETs and circulating markers of NETosis, respectively (356). Although metformin inhibited NETosis in vitro by reducing proteinase-3, histones and extracellular DNA, it did not affect insulin synthesis. In neutrophils, metformin prevented membrane translocation of PKCβII and activation of Nox, thereby decreasing NETosis in response to PMA and Ca²⁺. In line with this, metformin also decreased NET components in the plasma of patients with type 2 diabetes before and after treatment with insulin or dapagliflozin (357).

Circulating neutrophil levels are often increased in patients with a polycystic ovarian syndrome which is associated with an increased risk to develop ovarian cancer (358–360). Ibanez et al., reported that metformin can reduce neutrophil count in polycystic ovarian disease (360). These studies support the idea that metformin can prevent the increased neutrophil levels and NETosis that are associated with aggressive forms of ovarian cancer.

NET-independent mechanisms of metformin have also been observed. Metformin could impair tumor growth when administered during fasting-induced hypoglycemia. The anti-tumor effects of metformin were mediated by glycogen synthase kinase 3β (GSK3β) activation and PP2A-B56δ complex formation (361). Metformin inhibited the growth of a variety of breast cancer cells by inducing cell cycle arrest and apoptosis (362). Similar to other cell lines, metformin also induces AMPK activation, reduced the phosphorylation of epidermal growth factor receptor (EGFR), mitogen activated protein kinases (MAPKs) and Src and lowered the levels of cyclins D1 and E in breast cancer cells (362). Metformin also inhibits signal transducer and activator of transcription 3 (STAT3) activation and thereby reduced cell proliferation (363). Furthermore, metformin also activates p53 by activating AMPK, ultimately stopping the cell cycle (357, 364).

Metformin inhibits the proliferation of breast cancer cells with aberrant expression of human epidermal growth factor receptor 2 (HER2). Translational suppression of HER2 expression was observed after metformin treatment and this effect was triggered by the inhibition of the mTOR-S6K1 signaling pathway (365). Besides breast cancer, metformin can inhibit the proliferation of prostate, endometrial and brain cancer cells. Similar effects induced by metformin, inducing cell cycle arrest and suppressing the mTOR signaling pathway (366–368). In vivo experimental conditions, metformin treatment significantly reduced the primary tumor size of mammary adenocarcinomas and prolonged the lifespan of MMTV–Her2/Neu mice (369). Heterozygote mice of the tumor suppressor gene PTEN develop tumors in different organs, and metformin delayed tumor onset by 25% (370).

Many inhibitory effects of metformin on tumor growth through AMPK and mTOR signalings were confirmed using different mouse models of cancer (371–375). Metformin was also effective in reducing the growth of intestinal polyps in tumor suppressor Apc-mutant mice (376) by reducing mTOR/S6K/S6 signaling in the epithelium of the intestine. Of note, in this intestinal tumorigenesis model, tumor growth was shown to be associated with increased neutrophil infiltration and NETosis (130), raising the possibility that metformin may also inhibit NET-dependent tumor growth.

Although numerous preclinical, clinical and epidemiological studies proposed that metformin treatment inhibits tumor growth compared to other hypoglycemic treatments, it is still an open question whether metformin can be a potential candidate for the treatments of cancers predisposing tumor microenvironment to the release of ETs.

Thrombomodulin is an endothelial receptor, playing an important role in vascular homeostasis and regulation of coagulation. Thrombomodulin forms a complex with thrombin, thereby inactivating the coagulant activity of thrombin which activates protein C and thrombin activatable fibrinolysis inhibitor (TAFI) (377). Besides thrombin, thrombomodulin also regulates the inactivation of complement 3b (378). Recombinant thrombomodulin is given to the patients with disseminated intravascular coagulation, thereby protecting them from tissue injury (379). Recombinant thrombomodulin neutralizes damage-associated molecular patterns (DAMPs), including histones and HMGB1, inhibits aberrant activation of the complement system, protecting the endothelium (380). Using in vitro platelet-neutrophil coculture models, Shimomura et al., demonstrated that recombinant thrombomodulin may inhibit LPS-induced NETosis (381). Later, Helms et al. found that treatment of rats with recombinant thrombomodulin during septic shock limits excessive neutrophil activation and rescues a balanced coagulation and immunothrombosis response (382). This promising therapeutic tool would be important to follow in the future using mouse models of cancer.

Activated protein C (APC) is a serine protease with anti-coagulant and anti-inflammatory effector functions. Activation of the blood coagulation cascade by TF induces thrombin generation and the formation of a fibrin network. In addition, thrombin binds to thrombomodulin, and activates protein C in complex with endothelial protein C receptor (EPCR). Zymogen protein C is cleaved by thrombin to generate functionally active APC. After protein cleavage, APC forms a complex with protein S, and inactivates coagulation factors (Va, VIIIa) and as a negative feedback loop, inhibits thrombin generation (383). Therefore, long-term APC treatment could potentially increase the risk of bleeding complications (384). Besides this function, APC binds and activates PAR1 thereby enhancing vascular barrier integrity through sphingosine-1-phosphate receptor 1 (S1P1)-VE-cadherin signaling (385, 386).

The anti-inflammatory effects of APC involve the inhibition of neutrophil activation, NET formation and cell death. APC can effectively inhibit PI3K-PKC-dependent NET formation and this process is strongly dependent on the functional crosstalk between the macrophage-1 antigen (Mac-1), EPCR, and protease-activated receptor 3 (PAR3). APC can cleave PAR3 at a different site than thrombin, thereby inhibiting NET formation. Consequently, antibodies of EPCR, PAR3 and Mac-1 can reverse APC-mediated inhibition of NETosis (387).

Due to the multiple roles of APC in hemostasis and inflammation, it is difficult to predict the positive or negative effects of APC treatment in cancer progression. Increased levels of APC in the blood may limit metastasis by protecting the vascular barrier through VE-cadherin, but it may stimulate the metastatic potential of cancer cells (386). It has been shown that APC signaling enhances cancer cell migration, invasion and angiogenesis and also inhibits apoptosis (386, 388, 389). APC treatment could enhance breast cancer cell invasion in a dose-dependent manner (389). Cancer patients require long-term APC treatment which may induce severe bleeding complications (386), due to the hemostatic effects of APC on thrombin generation and factor Va/VIIIa functions. Although APC has a strong anti-inflammatory potential for the treatment of human patients, it is necessary to test recombinant mutant forms of APC with selective anti-inflammatory function in experimental models of cancer, without affecting thrombin generation and hemostasis.

Diphenyleneiodonium chloride (DPIC) is a hypoglycemic agent, identified as an inhibitor of NADH/Nox with highly potent anti-microbial activity against Mycobacterium tuberculosis and Staphylococcus aureus (390, 391). However, DPIC can also inhibit nitric oxide synthase (392), xanthine oxidase (393) and NADPH cytochrome P450 oxidoreductase (394), thereby inhibiting ROS production. Furthermore, DPIC inhibits oxidative phosphorylation (OXPHOS) and consequently reduces ATP production, thereby switching energy production to the lactic acid energy system (390). DPIC treatment strongly inhibits mitochondria function, thus leading to metabolic senescence (395). Interestingly, the effects of short-term DPIC treatment on cancer cells were independent of p53. However, long-term treatment showed that p53 expression facilitates a prolonged cell cycle arrest and protects cancer cells from apoptosis, while p53 deficiency could induce apoptosis with poly ADP-ribose polymerase (PARP) cleavage and DNA fragmentation in cancer cells (396). Altogether, these results suggest that DPIC treatment can reduces tumor growth by the inhibition of cancer cell proliferation and activation of the immune system through factors secreted by senescent cancer cells.

DPIC inhibits extracellular DNA release in PMA-stimulated neutrophils, although the degree of DPIC-inhibited NET formation was strongly dependent on the dose of external stimuli (49, 80). In different lung epithelial cells, NET formation is significantly increased by the secretion of CXCL8, IL8 and IL6 and this process was inhibited by DPIC (397). In the model of in vitro cigarette smoke extract-induced NETosis, DPIC treatment also inhibited this process (398). In mice, tobacco smoke increases lung metastasis by sustaining lung inflammation and thereby inducing NETosis which subsequently awake dormant cancer cells (108). Further investigation is necessary to show the effects of DPIC in long-term treated tumors and tumor microenvironment in vivo, focusing on the context of mitochondria dysfunction and senescence, as well as the distribution of NETs in cancer and lung metastasis.

High mobility group box-1 (HMGB1) is a nonhistone chromatin-associated protein, and as a nuclear cofactor in transcription regulation, interacting with many transcription factors and histones, supporting gene expression in the cells (399). However, HMGB1 is also secreted into the extracellular milieu, thereby initiating several interactions with receptors on the cell surface or with extruded DNA, triggering various signaling mechanisms and NETosis (399, 400). HMGB1 has a cytokine-like activity, thus regulating immune cell functions, including chemotaxis and immune modulation (401). In monocyte/macrophage-infiltrating disease conditions, HMGB1 facilitates macrophage reprogramming towards a proinflammatory phenotype through TLR4 activation (402). Interestingly, exposure to HMGB1 strongly increases the amount of extracellular DNA and citrullinated histone 3 in wild-type neutrophils, however, this effect was not observed in TLR4-deficient neutrophils (403). In mouse models treated with LPS, HMGB1 antibody treatment could decrease the levels of citrullination of histone 3 (403). Altogether, these results suggest that HMGB1 is a potential target for the development of anti-inflammatory therapies against TLR4-mediated NETosis.

Interestingly, metformin as a potential inhibitor of NETosis directly binds to the C-terminal tail of HMGB1 (357, 404). In the acute liver damage model, HMGB1 was released from damaged liver cells and metformin treatment could inhibit this process, protecting the liver cells (404). In another mouse model, metformin can significantly inhibit HMGB1 secretion and consequently reduce LPS-induced macrophage inflammatory responses, thereby improving the survival of endotoxemic mice (405). Altogether, these results suggest that metformin would be a potential drug to inhibit HMGB1-induced inflammation and NETosis.

Platelets are the major reservoirs of HMGB1, and it is released by activated platelets (400). HMGB1 binds TLR4 receptors on the platelet surface, thereby inducing recruitment of myeloid differentiation primary response 88 (Myd88) and guanylyl cyclase to the plasma membrane, leading to the activation of cGMP-dependent protein kinase I (400, 406). In a mouse model lacking HMGB1 in platelets, decreased thrombosis, lung inflammation and NETosis were observed indicating pleiotropic effects of HMGB1 in thromboinflammation (406).

Cancer cell-derived HMGB1 can modulate platelet-resident TLR4 receptors, thereby increasing platelet-dependent tumor metastasis. Although NETosis was not addressed in this study, blocking HMGB1 function in tumor cells was effective to inhibit tumor metastasis (407). HMGB1 is also expressed in keratinocytes. HMGB1-deficient keratinocytes displayed a marked reduction in NET formation, and subsequently delaying wound healing and promoting tumorigenesis in mice (408). Using anti-HMGB1 antibody treatment, HMGB1-mediated NETosis was strongly inhibited (403). It is tempting to investigate whether HGMB1 may also trigger DNA release from other inflammatory immune cells in proinflammatory tumor microenvironment.

Purinergic P2Y12 receptor (P2Y12) blockers (clopidogrel, ticagrelor, cangrelor, prasugrel) are widely used in patients with cerebrovascular, coronary artery, cerebrovascular and peripheral vascular diseases (409). The thienopyridine-derived metabolite irreversibly inhibits the binding of ADP to the receptor P2Y12, resulting in decreased platelet activation and aggregation responses, and reducing inside-out activation of platelet integrin αIIbβ3 integrin (410). Neutrophil-mediated platelet activation was suggested to be dependent on ADP (411, 412), therefore ADP blockers consequently attenuate platelet-neutrophil interactions and NETosis. In a mouse model of cholesterol crystal embolism, extracellular DNA has been exposed from NETs and damaged endothelium and activated platelets from emboli and vascular occlusion, leading to tissue infarction and kidney injury (231). In this model, P2Y12 blockade similarly to the DNAse I treatment strongly inhibited platelet function, consequently inhibiting extracellular DNA release and associated ischemia and organ injury (231).

In ST-elevation myocardial infarction (STEMI), the interaction between platelets and neutrophils results in the secretion of polyP in the presence of thrombin (413). In the infarct-related arteries, platelets release polyP, stimulating neutrophils to form thrombogenic/TF-bearing NETs (414, 415). Although ticagrelor significantly inhibited the NETotic effect of coronary stents in vitro, this did not rely on the P2Y12 receptor. These results indicated that ticagrelor may have pleiotropic effects on NETosis independently of platelets (415). The pancreatic cancer microenvironment is highly rich in tumor-associated neutrophils, platelets and NETs (416). Clopidogrel was shown to inhibit cancer growth and metastasis in PANC02 pancreatic cancer model (417). In the future, it will be important to analyse the effect of P2Y12 blockers on cancer-associated neutrophil activation and NETosis.

Disulfiram inhibits aldehyde dehydrogenase and is used to treat alcohol dependence (418). Disulfiram is also a potent inhibitor of gasdermin D in mouse and human macrophages and neutrophils (419, 420). Gasdermin D is a pore-forming protein playing a pivotal role in inflammatory cell death (419). In macrophages, inflammasome activation by canonical and or non-canonical pathways induces the cleavage of gasdermin D, which translocates to the plasma membrane thereby forming pores and inducing pyroptosis (421). In neutrophils, cytoplasmic caspase was shown to be directly activated by LPS or gram-negative bacteria independently of TLR4 (422). A recent study by Silva et al., showed that during sepsis caspase-11 activation induces gasdermin D cleavage, resulting in NET formation (420). Besides these mechanisms, gasdermin D cleavage is also generated by NE, which is released from neutrophils upon activation (423). Interestingly, inhibition of gasdermin D with disulfiram abolished NET formation reducing multiple organ dysfunction and sepsis-associated lethality (420). These studies indicate that disulfiram could be an important therapeutic agent to target gasdermin D, thereby preventing organ injury.

Diethylcarbamazine (DEC) is a derivate of piperazine, used as an anti-parasitic drug (424). Although at low doses DEC improves cytokine production, a high dose of this drug increases the respiratory burst in neutrophils (425). In vivo, DEC reduces the inflammatory granuloma formation in a bacterial infection model (426). DEC also decreases NET formation of neutrophils isolated from healthy subjects upon in vitro activation with PMA (427). In a follow-up study, DEC in both healthy donors and diabetes mellitus type 2 patients displayed an immunomodulatory effect inhibiting and delaying the tendency toward NET formation by their neutrophils (428). DEC in addition to inhibiting NETosis, also inhibits COX2, NF-kB activation, iNOS, TNFα and IL1β (429), indicating that the effects of DEC can be associated with many immunomodulatory pathways.

Glucuronoxylomannan (GXM), a polysaccharide, represents the main capsular content of the opportunistic yeast Cryptococcus neoformans, which has potent immunosuppressive properties. In a mouse model of rheumatoid arthritis triggered by collagen type II, GXM could improve the disease severity, by downregulating the cytokine and growth factor (TNFα, IL1β, IL6 and TGFβ) levels, thereby inhibiting Th17 cell differentiation and subsequent IL17 secretion (430). Furthermore, Rocha et al., showed that GXM treatment could abolish NET formation, independently of the agonist and stimuli (431). Future studies are required to validate whether GXM could be a potential therapeutic tool in ET-mediated thromboinflammation and cancer triggered by various cell types.

Anti-citrullinated protein antibodies (ACPAs), produced against citrullinated proteins, are diagnostic and prognostic markers of rheumatoid arthritis (432). Recent studies also provided evidence for circulating autoantibodies against citrullinated tumor-associated proteins in breast cancer patients (433). Anti-citrullinated proteins specifically targeting citrulline at histone 2A and 4 positions were proposed as a direct approach to inhibit murine and human NET formation (434).