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

Figures

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  FIGURE 1. Baseline values and the effects of ASA or L-ASA on eicosanoids of human serum generated by targeted chiral lipidomics analysis using LC-MS/MS. (A–C) Pie chart reporting the generation of eicosanoids in serum samples obtained in human whole blood allowed to clot for 1 h at 37 °C without vehicles (A) and in the presence of DMSO (ASA vehicle) (B) or diluent (triacetin and saccharin, the constituents of L-ASA) (C); data are the percentage of each eicosanoid versus the total concentration of all eicosanoids obtained by the mean of 6 separate experiments. (D,E) Heat map of the effect of ASA and L-ASA (0.01–300 μM), respectively, on eicosanoid (mean values of three separate experiments) biosynthesis in serum, respectively. As described in the Methods Section, eicosanoid levels were measured by LC-MS/MS.

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  FIGURE 2. Comparison of the effects of Aspirin and L-ASA on eicosanoids generated from COX-1 in whole blood. Aliquots (1 ml) of whole blood were collected from healthy volunteers and incubated with increasing concentrations of ASA (0.01–300 μM) or L-ASA (0.01–300 µM) or their vehicles (DMSO or diluent) for 1 h at 37°C. Serum was obtained by centrifugation, and 15S-HETE and 15R-HETE, PGE2, and TXB2 levels were measured by LC-MS/MS. (A,B) Heat map of the effect of ASA and L-ASA on eicosanoids biosynthesis in serum (mean values of three separate experiments); results are reported as % of inhibition from baseline values, in the presence of DMSO or diluent, respectively. (C) Concentration-response curve for inhibition of serum TXB2 by ASA and L-ASA using a validated and specific immunoassay; results are reported as mean ± SD (n = 4–7) of % inhibition from baseline values, in the presence of DMSO or diluent, respectively; concentration-response curves were fitted, and IC50 with 95% Confidence Interval (CI) values were reported.

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  FIGURE 3. Effects of ASA and L-ASA on the biosynthesis of TXB2 and the extent of acetylation of COX-1 at Serine529 in washed human platelets. (A)Washed platelet suspensions (0.5 ml containing 1 × 108 cells) were incubated with DMSO or diluent or ASA or L-ASA for 30 min at room temperature; then CaCl2 2 mM and MgSO4 1 mM were added for 2 min at room temperature, and platelets were stimulated with AA 10 μM for 30 min at 37 °C; finally, platelet suspensions were centrifuged, and supernatants were collected to assess TXB2 levels by immunoassay (validated by comparison with LC-MS/MS); results are reported as % inhibition from baseline values (mean ± SD, n = 4–6). (B) Washed human platelet suspensions were incubated for 60 min with DMSO or diluent or increasing concentrations of ASA or L-ASA at 37°C; then, platelets were lysed, and the quantification of acetylated and unacetylated COX-1 at Serine529 was assessed by LC-MS/MS, as described in materials and methods; the percentage of COX-1 acetylation to total COX-1 was calculated (%AceCOX-1). The data are reported as mean ± SD (n = 3 separate experiments). Concentration-response curves were fitted, and IC50 or EC50 values with 95% Confidence…

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  FIGURE 4. Biosynthesis of eicosanoids by human colon cancer HCA7 cells in response to physiological or high concentrations of exogenous AA in the presence of DMSO or diluent (triacetin and saccharin) by targeted chiral lipidomics analysis using LC-MS/MS. HCA7 cells (1 × 106) were treated with DMSO or diluent for 30 min, and then AA 0.5 (A,B), 10 (C,D), or 100 μM (E,F) was added, and the incubation was continued for further 30 min; then, prostanoids and HETEs were assessed in the conditioned medium by LC-MS/MS. Pie charts reporting the generation of eicosanoids in cancer cells from 4 separate experiments; data are shown as the percentage of each eicosanoid versus the total concentration of all eicosanoids produced in the presence of DMSO (ASA vehicle) or diluent (triacetin, saccharin, constituents of L-ASA).

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  FIGURE 5. The extent of COX-2 acetylation at Serine516 in HCA-7 cells by ASA and L-ASA. HCA-7 cells (3.0 × 106) were treated with DMSO, the diluent or increasing concentrations of ASA or L-ASA for 60 min at 37 °C. The cells were lysed, and the quantification of acetylated and unacetylated COX-2 was performed after protein digestion by LC-MS/MS as described in Materials and Methods. The percentage of COX-2 acetylation to total COX-2 was calculated (%AceCOX-2). Results are shown as mean ± SD, n = 3–4. Concentration-response curves were fitted, and IC50 or EC50 values with 95% Confidence Interval (CI) values are reported.

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  FIGURE 6. Effects of ASA and L-ASA on eicosanoid generation in response to physiological or high concentrations of AA in HCA7 cells. HCA-7 cells (1 × 106) were treated with DMSO or diluent or increasing concentrations of ASA or L-ASA for 30 min at 37°C, then AA 0.5 (A,B), 10 (C,D), or 100 μM (E,F) was added, and the incubation was continued for further 30 min. Prostanoids and HETEs were assessed in the conditioned medium by LC-MS/MS. A heat map of the effect of ASA and L-ASA on eicosanoids biosynthesis (mean values of 4 separate experiments) is shown; results are reported as ng/ml.

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  FIGURE 7. Concentration-response curves for inhibition of PGE2 in HCA7 cells in response to different concentrations of AA by ASA and L-ASA. The biosynthesis of PGE2 in HCA7 cells was evaluated by preincubating the cells (1 ×106) with vehicles (DMSO or diluent) or increasing concentrations of ASA or L-ASA for 30 min at 37°C; then, cells were incubated with AA 0.5 (A), 10 (B) or 100 µM (C) for 30 min at 37°C and the levels of PGE2 were assessed in the medium by LC-MS/MS. Data (% of inhibition from baseline with DMSO or diluent) are reported as mean ± SD, n = 4. Concentration-response curves were fitted, and IC50 values with their 95% CI values are shown.

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  FIGURE 8. Effects of ASA and L-ASA on the generation of 15R-HETE and 15S-HETE and other HETEs in HCA7 cells in response to AA 100 μM. HCA7 cells were treated with DMSO or diluent or increasing concentrations of the ASA (A) or L-ASA (B) for 30 min at 37°C; then, cells were incubated with AA100 µM for 30 min at 37°C, and the levels of 15S-HETE and 15R-HETE were assessed by LC-MS/MS; data are shown as ng/ml and reported as mean ± SEM, n = 4. The effects of ASA and L-ASA on other HETEs is shown in panel (C) and (D), respectively and reported as % of control (mean ± SEM, n = 4).