Platelets induce free and phospholipid-esterified 12-hydroxyeicosatetraenoic acid generation in colon cancer cells by delivering 12-lipoxygenase.

Platelets promote tumor metastasis by inducing promalignant phenotypes in cancer cells and directly contributing to cancer-related thrombotic complications. Platelet-derived extracellular vesicles (EVs) can promote epithelial-mesenchymal transition (EMT) in cancer cells, which confers high-grade malignancy. 12S-hydroxyeicosatetraenoic acid (12-HETE) generated by platelet-type 12-lipoxygenase (12-LOX) is considered a key modulator of cancer metastasis through unknown mechanisms. In platelets, 12-HETE can be esterified into plasma membrane phospholipids (PLs), which drive thrombosis. Using cocultures of human platelets and human colon adenocarcinoma cells (line HT29) and LC-MS/MS, we investigated the impact of platelets on cancer cell biosynthesis of 12S-HETE and its esterification into PLs and whether platelet ability to transfer its molecular cargo might play a role. To this aim, we performed coculture experiments with CFSE[5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester]-loaded platelets. HT29 cells did not generate 12S-HETE or express 12-LOX. However, they acquired the capacity to produce 12S-HETE mainly esterified in plasmalogen phospholipid forms following the uptake of platelet-derived medium-sized EVs (mEVs) expressing 12-LOX. 12-LOX was detected in plasma mEV of patients with adenomas/adenocarcinomas, implying their potential to deliver the protein to cancer cells in vivo. In cancer cells exposed to platelets, endogenous but not exogenous 12S-HETE contributed to changes in EMT gene expression, mitigated by three structurally unrelated 12-LOX inhibitors. In conclusion, we showed that platelets induce the generation of primarily esterified 12-HETE in colon cancer cells following mEV-mediated delivery of 12-LOX. The modification of cancer cell phospholipids by 12-HETE may functionally impact cancer cell biology and represent a novel target for anticancer agent development.

Multifaceted Functions of Platelets in Cancer: From Tumorigenesis to Liquid Biopsy Tool and Drug Delivery System.

Platelets contribute to several types of cancer through plenty of mechanisms. Upon activation, platelets release many molecules, including growth and angiogenic factors, lipids, and extracellular vesicles, and activate numerous cell types, including vascular and immune cells, fibroblasts, and cancer cells. Hence, platelets are a crucial component of cell-cell communication. In particular, their interaction with cancer cells can enhance their malignancy and facilitate the invasion and colonization of distant organs. These findings suggest the use of antiplatelet agents to restrain cancer development and progression. Another peculiarity of platelets is their capability to uptake proteins and transcripts from the circulation. Thus, cancer-patient platelets show specific proteomic and transcriptomic expression patterns, a phenomenon called tumor-educated platelets (TEP). The transcriptomic/proteomic profile of platelets can provide information for the early detection of cancer and disease monitoring. Platelet ability to interact with tumor cells and transfer their molecular cargo has been exploited to design platelet-mediated drug delivery systems to enhance the efficacy and reduce toxicity often associated with traditional chemotherapy. Platelets are extraordinary cells with many functions whose exploitation will improve cancer diagnosis and treatment.

Platelet-Specific Deletion of Cyclooxygenase-1 Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice.

Inflammatory bowel disease (IBD) is associated with an increased risk for thromboembolism, platelet activation, and abnormalities in platelet number and size. In colitis, platelets can extravasate into the colonic interstitium. We generated a mouse with a specific deletion of cyclooxygenase (COX)-1 in megakaryocytes/platelets [(COX-1 conditional knockout (cKO)] to clarify the role of platelet activation in the development of inflammation and fibrosis in dextran sodium sulfate (DSS)-induced colitis. The disease activity index was assessed, and colonic specimens were evaluated for histologic features of epithelial barrier damage, inflammation, and fibrosis. Cocultures of platelets and myofibroblasts were performed. We found that the specific deletion of COX-1 in platelets, which recapitulated the human pharmacodynamics of low-dose aspirin, that is, suppression of platelet thromboxane (TX)A2 production associated with substantial sparing of the systemic production of prostacyclin, resulted in milder symptoms of colitis, in the acute phase, and almost complete recovery from the disease after DSS withdrawal. Reduced colonic accumulation of macrophages and myofibroblasts and collagen deposition was found. Platelet-derived TXA2 enhanced the ability of myofibroblasts to proliferate and migrate in vitro, and these effects were prevented by platelet COX-1 inhibition or antagonism of the TXA2 receptor. Our findings allow a significant advance in the knowledge of the role of platelet-derived TXA2 in the development of colitis and fibrosis in response to intestinal damage and provide the rationale to investigate the potential efficacy of the antiplatelet agent low-dose aspirin in limiting the inflammatory response and fibrosis associated with IBD. SIGNIFICANCE STATEMENT: Inflammatory bowel disease (IBD) is characterized by the development of a chronic inflammatory response, which can lead to intestinal fibrosis for which currently there is no medical treatment. Through the generation of a mouse with specific deletion of cyclooxygenase-1 in megakaryocytes/platelets, which recapitulates the human pharmacodynamics of low-dose aspirin, we demonstrate the important role of platelet-derived thromboxane A2 in the development of experimental colitis and fibrosis, thus providing the rationale to investigate the potential efficacy of low-dose aspirin in limiting the inflammation and tissue damage associated with IBD.

Platelets in cancer development and diagnosis.

Platelets are involved in the development and progression of cancer through several mechanisms. Platelet activation at the site of tissue damage contributes to the initiation of a cascade of events which promote tumorigenesis. In fact, platelets release a wide array of proteins, including growth and angiogenic factors, lipids and extracellular vesicles rich in genetic material, which can mediate the induction of phenotypic changes in target cells, such as immune, stromal and tumor cells, and promote carcinogenesis and metastasis formation. Importantly, the role of platelets in tumor immune escape has been described. These lines of evidence open the way to novel strategies to fight cancer based on the use of antiplatelet agents. In addition to their ability to release factors, platelets are able of up-taking proteins and genetic material present in the bloodstream. Platelets are like 'sentinels' of the disease state. The evaluation of proteomics and transcriptomics signature of platelets and platelet-derived microparticles could represent a new strategy for the development of biomarkers for early cancer detection and/or therapeutic drug monitoring in cancer chemotherapy. Owing to the ability of platelets to interact with cancer cells and to deliver their cargo, platelets have been proposed as a 'biomimetic drug delivery system' for anti-tumor drugs to prevent the occurrence of off-target adverse events associated with the use of traditional chemotherapy.

Tumor-Educated Platelet Extracellular Vesicles: Proteomic Profiling and Crosstalk with Colorectal Cancer Cells.

BACKGROUND: Platelet-cancer cell interactions modulate tumor metastasis and thrombosis in cancer. Platelet-derived extracellular vesicles (EVs) can contribute to these outcomes. METHODS: We characterized the medium-sized EVs (mEVs) released by thrombin-stimulated platelets of colorectal cancer (CRC) patients and healthy subjects (HS) on the capacity to induce epithelial-mesenchymal transition (EMT)-related genes and cyclooxygenase (COX)-2(PTGS2), and thromboxane (TX)B2 production in cocultures with four colorectal cancer cell lines. Platelet-derived mEVs were assessed for their size distribution and proteomics signature. RESULTS: The mEV population released from thrombin-activated platelets of CRC patients had a different size distribution vs. HS. Platelet-derived mEVs from CRC patients, but not from HS, upregulated EMT marker genes, such as TWIST1 and VIM, and downregulated CDH1. PTGS2 was also upregulated. In cocultures of platelet-derived mEVs with cancer cells, TXB2 generation was enhanced. The proteomics profile of mEVs released from activated platelets of CRC patients revealed that 119 proteins were downregulated and 89 upregulated vs. HS. CONCLUSIONS: We show that mEVs released from thrombin-activated platelets of CRC patients have distinct features (size distribution and proteomics cargo) vs. HS and promote prometastatic and prothrombotic phenotypes in cancer cells. The analysis of platelet-derived mEVs from CRC patients could provide valuable information for developing an appropriate treatment plan.

Pharmacological characterization of the biosynthesis of prostanoids and hydroxyeicosatetraenoic acids in human whole blood and platelets by targeted chiral lipidomics analysis.

Platelet 12-lipoxygenase(p-12-LOX) is highly expressed in human platelets, and the development of p-12-LOX inhibitors has the potential to be a novel antithrombotic tool by inhibiting thrombosis without prolonging hemostasis. A chiral liquid chromatography-mass spectrometry(LC-MS/MS) method was used to assess the impact of three commercially available LOX inhibitors[esculetin(6,7-dihydroxycoumarin), ML-355(N-2-benzothiazolyl-4-[[(2-hydroxy-3-methoxyphenyl)methyl]amino]-benzenesulfonamide), CDC(cinnamyl-3,4-dihydroxy-α-cyanocinnamate) and acetylsalicylic acid(ASA; a cyclooxygenase-1 inhibitor) on the generation of prostanoids and HETEs(hydroxyeicosatetraenoic acids) in human whole blood allowed to clot for 1 h at 37 °C(serum), platelet-rich plasma(PRP) stimulated with collagen or TRAP-6(a peptide activating thrombin receptor) and washed platelets. In serum, ML-355 did not affect eicosanoid generation, while CDC caused an incomplete reduction of 12S-HETE levels; esculetin inhibited both 12S-HETE and thromboxane(TX)B2 production; ASA selectively affected TXB2 production. In washed platelets stimulated with thrombin, esculetin, and CDC inhibited both 12S-HETE and TXB2 while ML-355 was almost ineffective. In PRP, ML-355, CDC, and esculetin did not affect platelet aggregation associated with incomplete effects on eicosanoid biosynthesis. ASA alone or in combination with ticagrelor(a P2Y12 blocker) affected platelet aggregation associated with profound inhibition of TXB2 generation. P2Y12 receptor signaling contributed to platelet 12S-HETE biosynthesis in response to primary agonists. In conclusion, ML-355, esculetin, and CDC were not selective inhibitors of p-12-LOX in different cellular systems. They did not affect platelet aggregation induced in PRP by collagen or TRAP-6. The characterization of 12-LOX inhibitors on eicosanoids generated in human whole blood is useful for information on their enzyme selectivity, off-target effects, and the possible influence of plasma components on their pharmacological effects.

Characterization of cyclooxygenase-2 acetylation and prostanoid inhibition by aspirin in cellular systems.

The most recognized mechanism of aspirin (acetylsalicylic acid, ASA) action, at therapeutic dosing, is the inhibition of prostanoid biosynthesis through the acetylation of cyclooxygenase (COX)-isozymes (COX-1 at serine-529 and COX-2 at serine-516). Whether aspirin, also when given at the low-doses recommended for cardiovascular prevention, reduces the risk of colorectal cancer by affecting COX-2 activity in colorectal adenomatous lesions is still debated. We aimed to develop a direct biomarker of aspirin action on COX-2 by assessing the extent of acetylation of COX-2 at serine-516 using the AQUA strategy, enabling absolute protein quantitation by liquid chromatography-mass spectrometry. We compared the extent of acetylation and the inhibition of prostanoid biosynthesis by ASA using human recombinant COX-2 (hu-COX-2), the human colon cancer cell line HCA-7, isolated human monocytes stimulated with LPS (lipopolysaccharide) or human intestinal epithelial cells stimulated with interleukin (IL)-1ß. Hu-COX-2 exposed in vitro to an excess of ASA was acetylated by approximately 40-50% associated with the inhibition of COX-2 activity by 80-90%. In the three cell-types expressing COX-2, the extent of COX-2 acetylation and reduction of prostaglandin (PG) E2 biosynthesis by ASA was concentration-dependent with comparable EC50 values (in the low µM range). The maximal % acetylation of COX-2 averaged 80%, at ASA 1000 µM, and was associated with a virtually complete reduction of PGE2 biosynthesis (97%). In conclusion, we have developed a proteomic assay to evaluate the extent of acetylation of COX-2 at serine-516 by aspirin; its use in clinical studies will allow clarifying the mechanism of action of aspirin as anticancer agent.