Inhibition of activator protein 1 activity and neoplastic transformation by aspirin

Abstract

Aspirin, along with its analgesic-antipyretic uses, is now also being considered for prevention of cardiovascular disease, cancer, and treatment of human immunodeficiency virus infection. Although many of aspirin's pharmacological actions are related to its ability to inhibit prostaglandin biosynthesis, some of its beneficial therapeutic effects are not completely understood. Transcription factor activator protein 1 (AP-1) is critical for the induction of neoplastic transformation and induction of multiple genes involved in inflammation and infection. We have used the JB6 mouse epidermal cell lines, a system that has been used extensively as an in vitro model for the study of tumor promotion and anti-tumor promotion, to study the anti-carcinogenesis effect of aspirin at the molecular level. Aspirin and aspirin-like salicylates inhibited the activation of AP-1 in the same dose range as seen for the inhibition of tumor promoter-induced transformation. The inhibition of AP-1 and tumor promoter-induced transformation in JB6 cells occurs through a prostaglandin independent- and an Erk1- or Erk2-independent pathway. The mechanism of AP-1 and transformation inhibition in this cell culture model may involve the elevation of H+ concentration. The inhibition effects on the activation of AP-1 activity by aspirin and aspirin-like salicylates may further explain the anti-carcinogenesis mechanism of action of these drugs.

Fig. 1. Inhibition of TPA-induced transformation by aspirin and SA

10⁴ JB6 P⁺ cells (Cl41-19) were exposed simultaneously to Me₂SO (0.01%, control) or TPA with or without aspirin (A) or SA (B) in 0.33% agar for 14 days and scored for colonies at the end of the experiment. Results are expressed as the mean of three independent experiments ± standard error.

Fig. 2. The effects of aspirin or SA on [³H]TdR incorporation into cells DNA

5 × 10³ JB6 Cl41-19 cells were exposed to aspirin or SA for 36 h. Then [³H]TdR (0.5 μCi/well) was added to each well. The cells were harvested 12 h later, and incorporation of [³H]TdR was detected.

Fig. 3. Inhibition of AP-1 activity of JB6 P⁺ cells by aspirin and SA

Stable AP-1-Luc transfectants Cl41-19 cells were exposed to TPA with or without aspirin (A) or SA (B) at different concentrations for 24 h. Results are expressed as the mean of three independent experiments ± standard error.

Fig. 4. Inhibition of TIMP-1 mRNA expression by aspirin and SA

RNA from Cl41-19 cells exposed with or without aspirin or SA for 24 h was analyzed by Northern blots.

Fig. 5. Lack of effects on TPA-induced AP-1 activity or transformation by indomethacin

For assaying AP-1 activity (A), JB6 Cl41-19 cells were exposed to 0.01% of Me₂SO (the solvent control group) or TPA or different concentrations of indomethacin for 24 h. The AP-1 luciferase enzyme activity was measured using the luminometer. For measuring transformation activity (B), 10⁴ JB6 P⁺ cells (Cl41-19) were exposed simultaneously to Me₂SO (0.01%, control) or TPA with or without indomethacin in 0.33% agar and scored for colonies at 14 days.

Fig. 6. Inhibition of prostaglandin E₂ synthesis by indomethacin

5 × 10⁴ P+ cells were seeded in each well of 6-well plates and cultured overnight. Then the cells were washed with serum-free medium and changed to 2 ml of serum-free medium with or without indomethacin or TPA. After 3 days culture, the medium was aspirated from each well for prostaglandin E enzyme immunoassays with a prostaglandin E₂ EIA Assay Kit (PerSeptive Diagnostics, Cambridge, MA). The results are expressed as the mean of three independent experiments ± standard error.

Fig. 7. Aspirin or SA does not inhibit basal or TPA-induced Erk1 or Erk2 or phosphorylated c-jun

JB6 Cl41-19 cells were pretreated with aspirin, SA, or medium alone for 24 h and then exposed to TPA with or without aspirin or SA for 30 min (A and B) or 24 h (C and D). The cells were lysed and Erk1 and Erk2 proteins and phosphorylation proteins were assayed by a PhosphoPlus MAPKs kit from New England Biolabs. For measuring the phosphorylation level of c-Jun protein (E and F), protein extracts from the same samples as described in C were assayed by a PhosphoPlus c-Jun kit from New England Biolabs.

Fig. 8. DES inhibits TPA-induced AP-1 activity and transformation

For assaying AP-1 activity (A), JB6 Cl41-19 cells were exposed to 0.01% of Me₂SO (the solvent control group) or TPA or different concentrations of DES for 24 h. The luciferase enzyme activity was measured using the luminometer. For assaying transformation activity (B), 10⁴ JB6 Cl41-19 cells were exposed to TPA with or without DES in 0.33% agar for 14 days. The results are expressed as the mean of three independent experiments ± standard error.

Fig. 9. The effects of DES on [³H]TdR incorporation into cells DNA

5 × 10³ JB6 Cl41-19 cells were exposed to DES for 36 h. Then [ ³H]TdR (0.5 μCi/well) was added to each well. The cells were harvested 12 h later, and incorporation of [³H]TdR was detected.