Suppression of aromatase in human breast cells by a cyclooxygenase-2 inhibitor and its analog involves multiple mechanisms independent of cyclooxygenase-2 inhibition

Abstract

Previous studies have demonstrated that cyclooxygenase-2 (COX-2) inhibitor NS-398 decrease aromatase activity at the transcript level in breast cancer cells. However, N-Methyl NS-398, which does not have COX-2 inhibitory activity but has very similar structure to NS-398, decreases aromatase activity and transcription in MCF-7 and MDA-MB-231 breast cells to the same extent as NS-398. This suggests that NS-398 decrease aromatase expression in breast cancer cells via other mechanism(s). Further investigations find that both compounds only decrease aromatase activity stimulated by forskolin/phorbol ester at the transcript level in both breast cancer cell lines and in breast stromal cells from patients. They do not affect aromatase expression and activity stimulated by dexamethasone. Both compounds also suppress MCF-7 cell proliferation stimulated by testosterone. Aromatase inhibition studies using placental microsomes demonstrate that the compounds show only weak direct aromatase inhibition. These results suggest that NS-398 and its N-methyl analog suppress aromatase expression and activity with multiple mechanisms.

Figure 1

Structure of NS-398 and N-Methyl NS-398 and their COX-2 inhibition results in MDA-MB-231 cell line [28].

Figure 2. Suppression of aromatase activity in MCF-7 and MDA-MB-231 breast cancer cells

Cells were treated with NS-398 and MNS-398 at indicated concentrations. The results were normalized against a control treatment with vehicle. Each data bar represents the mean results of three independent determinations. (A) MCF-7 cells, the value of 100% is equal to 0.0002 pmol/hr/10⁶ cells. (B) MDA-MB-231 cells, the value of 100% is equal to 0.00007 pmol/hr/10⁶ cells. *P <0.05 vs. control by unpaired t test.

Figure 3. Aromatase inhibition of microsomal activities of compounds NS-398 (■) and Me-NS-398 (▲)

Error bars represent standard error (n = 3), and the data were statistically analyzed by a nonlinear regression analysis method.

Figure 4. Real-time RT-PCR analysis of CYP19 mRNA expression in MCF-7 (A) and MDA-MB-231 cells (B)

Cells were treated for 24 h with the test agents at different concentrations, and total RNA was isolated. Results are expressed as means of CYP19 (normalized to 18S rRNA) ± SEM. *P <0.05 vs. control by unpaired t test (n = 9).

Figure 5. Effect of NS-398 and Me-NS-398 on induced aromatase in breast cancer cells

Cells were pre-treated with FSK(25μM)/TPA (10nM) or DEX(200nM) for 24 hr, then with the indicated compounds (2μM), Ru486(200nM) and FSK/TPA or DEX for 24 hr. Aromatase activity was subsequently determined during a 6 h assay as described. *p<0.05 vs FSK/TPA or DEX treatment for suppression of induced aromatase activity; **p<0.05 vs DMSO treatment for stimulation of aromatase activity by unpaired t test (n=3). (A) MCF-7 cells, the value of 100% is equal to 0.0002pmol/10⁶ cells/hr. (B) MDA-MB-231 cells, the value of 100% is equal to 0.00007pmol/10⁶ cells/hr. Similar results were obtained in at least two independent experiments.

Figure 6. Effect of NS-398 and Me-NS-398 on induced CYP19 gene expression in MCF-7 breast cancer cells

Cells were pre-treated with FSK(25μM)/TPA (10nM) for 24 hr, then with the indicated compounds (2μM) and FSK/TPA for 24 hr. Total RNA was isolated. Results are expressed as means of CYP19 (normalized to 18S rRNA) ± SEM., *p<0.05 vs FSK/TPA treatment for suppression of induced aromatase transcription; **p<0.05 vs DMSO treatment for stimulation of aromatase transcription by unpaired t test (n = 9). Similar results were obtained in at least two independent experiments.

Figure 7. Effect of NS-398 and Me-NS-398 on induced aromatase in breast stromal cells

Cells were pre-treated with FSK(10μM)/TPA (10nM) or DEX(200nM) for 24 hr, then with the indicated compounds (2 μM), Ru486(200nM) and FSK/TPA or DEX for 24 hr. Aromatase activity was subsequently determined during a 6 h assay as described. *p<0.05 vs FSK/TPA or DEX treatment for suppression of induced aromatase activity; **p<0.05 vs DMSO treatment for stimulation of aromatase activity by unpaired t test (n = 3). The value of 100% is equal to 0.00012pmol/10⁶ cells/hr. Similar results were obtained in at least two independent experiments.

Figure 8. Effect of NS-398 and Me-NS-398 on induced CYP19 gene expression in breast stromal cells

Cells were pre-treated with FSK (10μM)/TPA (10nM) for 24 hr, then with the indicated compounds (2μM) and FSK/TPA for 24 hr. Total RNA was isolated. Results are expressed as means of CYP19 (normalized to 18S rRNA) ± SEM. *p<0.05 vs FSK/TPA treatment for suppression of induced aromatase transcription; **p<0.05 vs DMSO treatment for stimulation of aromatase transcription by unpaired t test (n=9). Similar results were obtained in at least two independent experiments.

Figure 9. Antiproliferation effect of NS-398 and Me-NS-398 in MCF-7 cells

MCF-7 cells were seeded into 24 wells plates at ~1×10⁴/well and exposed to testosterone (T, 100nM) or the test agent (NS-398 and Me-NS-398at 2 μM) plus testosterone (100nM) in DMEM/F12 media supplemented 5% charcoal stripped fetal bovine serum. Cell viability was measured as described in the experimental section. The results were normalized against a control treatment with vehicle (DMSO). *p<0.05 vs testosterone treatment for suppression of induced proliferation; **p<0.05 vs control for stimulation of proliferation by unpaired t test (n=6). Similar results were obtained in at least two independent experiments.