The specific deletion of cyclooxygenase-1 in megakaryocytes/platelets reduces intestinal polyposis in ApcMin/+ mice.

Clinical and experimental evidence sustain the role of cyclooxygenase (COX)-1 in intestinal tumorigenesis. However, the cell type expressing the enzyme involved and molecular mechanism(s) have not been clarified yet. We aimed to elucidate the role of platelet COX-1 (the target of low-dose aspirin in humans) in intestinal tumorigenesis of ApcMin/+ mice, considered a clinically relevant model. To realize this objective, we generated an ApcMin/+ mouse with a specific deletion of Ptgs1(COX-1 gene name) in megakaryocytes/platelets (ApcMin/+;pPtgs1-/-mice) characterized by profound inhibition of thromboxane(TX)A2 biosynthesis ex vivo (serum TXB2; by 99%) and in vivo [urinary 2,3-dinor-TXB2(TXM), by 79%]. ApcMin/+ mice with the deletion of platelet COX-1 showed a significantly reduced number (67%) and size (32%) of tumors in the small intestine. The intestinal adenomas of these mice had decreased proliferative index associated with reduced COX-2 expression and systemic prostaglandin(PG)E2 biosynthesis (urinary PGEM) vs. ApcMin/+mice. Extravasated platelets were detected in the intestine of ApcMin/+mice. Thus, we explored their contribution to COX-2 induction in fibroblasts, considered the primary polyp cell type expressing the protein. In the coculture of human platelets and myofibroblasts, platelet-derived TXA2 was involved in the induction of COX-2-dependent PGE2 in myofibroblasts since it was prevented by the selective inhibition of platelet COX-1 by aspirin or by a specific antagonist of TXA2 receptors. In conclusion, our results support the platelet hypothesis of intestinal tumorigenesis and provide experimental evidence that selective inhibition of platelet COX-1 can mitigate early events of intestinal tumorigenesis by restraining COX-2 induction.

Optimizing aspirin dose for colorectal cancer patients through deep phenotyping using novel biomarkers of drug action.

Background: Low-dose aspirin's mechanism of action for preventing colorectal cancer (CRC) is still debated, and the optimal dose remains uncertain. We aimed to optimize the aspirin dose for cancer prevention in CRC patients through deep phenotyping using innovative biomarkers for aspirin's action. Methods: We conducted a Phase II, open-label clinical trial in 34 CRC patients of both sexes randomized to receive enteric-coated aspirin 100 mg/d, 100 mg/BID, or 300 mg/d for 3 ± 1 weeks. Biomarkers were evaluated in blood, urine, and colorectal biopsies at baseline and after dosing with aspirin. Novel biomarkers of aspirin action were assessed in platelets and colorectal tissues using liquid chromatography-mass spectrometry to quantify the extent of cyclooxygenase (COX)-1 and COX-2 acetylation at Serine 529 and Serine 516, respectively. Results: All aspirin doses caused comparable % acetylation of platelet COX-1 at Serine 529 associated with similar profound inhibition of platelet-dependent thromboxane (TX)A2 generation ex vivo (serum TXB2) and in vivo (urinary TXM). TXB2 was significantly reduced in CRC tissue by aspirin 300 mg/d and 100 mg/BID, associated with comparable % acetylation of COX-1. Differently, 100 mg/day showed a lower % acetylation of COX-1 in CRC tissue and no significant reduction of TXB2. Prostaglandin (PG)E2 biosynthesis in colorectal tumors and in vivo (urinary PGEM) remained unaffected by any dose of aspirin associated with the variable and low extent of COX-2 acetylation at Serine 516 in tumor tissue. Increased expression of tumor-promoting genes like VIM (vimentin) and TWIST1 (Twist Family BHLH Transcription Factor 1) vs. baseline was detected with 100 mg/d of aspirin but not with the other two higher doses. Conclusion: In CRC patients, aspirin 300 mg/d or 100 mg/BID had comparable antiplatelet effects to aspirin 100 mg/d, indicating similar inhibition of the platelet's contribution to cancer. However, aspirin 300 mg/d and 100 mg/BID can have additional anticancer effects by inhibiting cancerous tissue's TXA2 biosynthesis associated with a restraining impact on tumor-promoting gene expression. EUDRACT number: 2018-002101-65. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT03957902.

Characterization of the acetylation of cyclooxygenase-isozymes and targeted lipidomics of eicosanoids in serum and colon cancer cells by the new aspirin formulation IP1867B versus aspirin in vitro.

Background: Aspirin(acetylsalicylic acid, ASA) is recommended for the secondary prevention of atherothrombotic events and has shown anticancer effects. The current enteric-coated drug formulation may reduce aspirin bioavailability. Liquid formulations could improve aspirin pharmacokinetics and pharmacodynamics. IP1867B is a liquid-aspirin formulation that combines three ingredients, ASA/triacetin/saccharin. Methods: ASA and IP1867B(L-ASA) were assessed in human serum(obtained by allowing to clot human whole blood at 37 °C for 1h), washed platelets, and colonic adenocarcinoma HCA7 cells on eicosanoid generation and COX-isozyme acetylation at Serine529 and 516 by LC-MS/MS. Results: In serum, ASA and L-ASA acted by selectively affecting COX-1-derived eicosanoids, including thromboxane(TX)B2. L-ASA was more potent in inhibiting serum TXB2, a known biomarker of aspirin antiplatelet effect, than ASA. However, ASA and L-ASA were equipotent to acetylate COX-1 in washed platelets and COX-2 in HCA7 cells. In HCA7 cells, ASA and L-ASA acted by inhibiting prostaglandin(PG)E2(the most abundant prostanoid) and TXB2 biosynthesis. In the presence of a high arachidonic acid concentration(100 µM), 15R-hydroxyeicosatetraenoic acid(HETE) was generated at baseline by cancer cell COX-2 and was only slightly enhanced by supratherapeutic concentrations of ASA(1 mM). In whole blood and HCA7 cells treated with ASA or L-ASA, 15-epi-lipoxin(LX)A4 were undetectable. Conclusion: IP1867B was more potent in affecting serum TXB2 generation than ASA. The relevance of this finding deserves evaluation in vivo in humans. In cancer cells, ASA and IP1867B acted by inhibiting PGE2 and TXB2 generation via the acetylation of COX-2. ASA and IP867B at clinically relevant concentrations did not substantially induce the biosynthesis of 15R-HETE and 15-epi-LXA4.

Biology and pharmacology of platelet-type 12-lipoxygenase in platelets, cancer cells, and their crosstalk.

Platelet-type lipoxygenase (pl12-LOX), encoded by ALOX12, catalyzes the production of the lipid mediator 12S-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12S-HpETE), which is quickly reduced by cellular peroxidases to form 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12S-HETE). Platelets express high levels of pl12-LOX and generate considerable amounts of 12S-HETE from arachidonic acid (AA; C20:4, n-6). The development of sensitive chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods has allowed the accurate quantification of 12S-HETE in biological samples. Moreover, advances in the knowledge of the mechanism of action of 12S-HETE have been achieved. The orphan G-protein-coupled receptor 31 (GPR31) has been identified as the high-affinity 12S-HETE receptor. Moreover, upon platelet activation, 12S-HETE is produced, and significant amounts are found esterified to membrane phospholipids (PLs), such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC), promoting thrombin generation. Platelets play many roles in cancer metastasis. Among them, the platelets' ability to interact with cancer cells and transfer platelet molecules by the release of extracellular vesicles (EVs) is noteworthy. Recently, it was found that platelets induce epithelial-mesenchymal transition(EMT) in cancer cells, a phenomenon known to confer high-grade malignancy, through the transfer of pl12-LOX contained in platelet-derived EVs. These cancer cells now generate 12-HETE, considered a key modulator of cancer metastasis. Interestingly, 12-HETE was mainly found esterified in plasmalogen phospholipids of cancer cells. This review summarizes the current knowledge on the regulation and functions of pl12-LOX in platelets and cancer cells and their crosstalk.Novel approaches to preventing cancer and metastasis by the pharmacological inhibition of pl12-LOX and the internalization of mEVs are discussed.