Positional isomers of aspirin are equally potent in inhibiting colon cancer cell growth: differences in mode of cyclooxygenase inhibition

Abstract

We compared the differential effects of positional isomers of acetylsalicylic acid (o-ASA, m-ASA, and p-ASA) on cyclooxygenase (COX) inhibition, gastric prostaglandin E2 (PGE2), malondialdehyde, tumor necrosis factor-alpha (TNF-α) levels, superoxide dismutase (SOD) activity, human adenocarcinoma colon cancer cell growth inhibition, cell proliferation, apoptosis, and cell-cycle progression. We also evaluated the gastric toxicity exerted by ASA isomers. All ASA isomers inhibit COX enzymes, but only the o-ASA exerted an irreversible inhibitory profile. We did not observe a significant difference between ASA isomers in their ability to decrease the in vivo synthesis of PGE2 and SOD activity. Furthermore, all isomers increased the levels of gastric and TNF-α when administered orally at equimolar doses. We observed a dose-dependent cell growth inhibitory effect; the order of potency was p-ASA > m-ASA ≈ o-ASA. There was a dose-dependent decrease in cell proliferation and an increase in apoptosis, with a concomitant Go/G1 arrest. The ulcerogenic profile of the three ASA isomers showed a significant difference between o-ASA (aspirin) and its two positional isomers when administered orally at equimolar doses (1 mmol/kg); the ulcer index (UI) for o-ASA indicated extensive mucosal injury (UI = 38), whereas m-ASA and p-ASA produced a significantly decreased toxic response (UI = 12 and 8, respectively) under the same experimental conditions. These results suggest that the three positional isomers of ASA exert practically the same biologic profile in vitro and in vivo but showed different safety profiles. The mechanism of gastric ulcer formation exerted by aspirin and its two isomers warrants a more detailed and thorough investigation.

Fig. 1.

Chemical structures of positional isomers of aspirin.

Fig. 2.

Effects of positional isomers of aspirin on gastric PGE₂ level, lipid peroxidation (MDA), SOD, and plasma TNF-α. Four groups of rats were treated with vehicle, o-ASA, m-ASA, and p-ASA, and their stomachs were removed and processed as described in Materials and Methods. All three drugs caused a significant reduction in gastric mucosal PGE₂ levels (A). Results are mean ± S.E.M. of five rats in each group; *P < 0.01 versus vehicle group. All three compounds caused an almost 6-fold increase in MDA levels (B). Results are mean ± S.E.M. for five rats in each group; *P < 0.01 versus vehicle group. All three compounds caused a significant reduction in SOD activity (C). Results are mean ± S.E.M. of five rats; *P < 0.05 versus vehicle group. All three compounds caused a significant rise in plasma TNF-α (D). Results are mean ± S.E.M. for three rats in each group; *P < 0.01 versus vehicle.

Fig. 3.

Positional isomers of aspirin inhibit the growth of HT-29 human colon cancer cells. Cells were treated with various concentrations of o- m- p- ASA as described in Materials and Methods. Cell numbers were determined at 24 hours. The IC₅₀ for cell growth inhibition was >5 mM for all three positional isomers of ASA; however, p-ASA was significantly more potent than the o- and m- isomer at all concentrations. Results represent means ± S.E.M. of three different experiments performed in triplicate. *P < 0.05 versus o-ASA and m-ASA.

Fig. 4.

Positional isomers of aspirin induce apoptosis and inhibit proliferation of HT-29 cells. Cells were treated with o-ASA, m-ASA, or p-ASA at the concentration indicated for 24 hours, after which the cells were stained with annexin and propidium iodide and subjected to flow cytometric analysis as described in Materials and Methods. The percentage of apoptotic cells increased in a concentration-dependent manner (A). Cells were treated with o-ASA, m-ASA, or p-ASA at the concentrations indicated for 24 hours, after which PCNA expression was determined by flow cytometry and expressed as a percentage positive cells as described in Materials and Methods (B). Results are mean ± S.E.M. of three different experiments. *P < 0.05 compared with untreated cells.

Fig. 5.

Effects of positional isomers of aspirin on cell cycle in HT-29 cells. Cells were treated for 24 hours with various concentrations of o-ASA, m-ASA, or p-ASA, and their cell-cycle phase distribution was determined by flow cytometry as described in Materials and Methods. Results are representative of two different experiments. This study was repeated twice, generating results within 10% of those presented here.

Fig. 6.

m-ASA and p-ASA cause less gastric damage compared with o-ASA. Drugs were administered orally at equimolar doses (1 mmol/kg) and effects on the stomach were evaluated as indicated in Materials and Methods. o-ASA caused severe gastric damage, UI = 45 ± 3.1 mm, whereas both m-ASA and p-ASA were relatively gastric damage sparing, UI = 14 ± 3.0 mm and 17 ± 4.1 mm for m-ASA and p-ASA, respectively (A). All three drugs also caused erosions of the gastric mucosa, but the damage was less with o-ASA compared with that of m-ASA and p-ASA (B). Results are mean ± S.E.M. for five rats in each group, *P < 0.05 compared with the vehicle group; ^†P < 0.05 compared with o-ASA.

Fig. 7.

Docking of positional isomers of aspirin to the active site of cyclooxygenase-1 and -2. Hydrogen atoms are not shown for clarity.