Berberine suppresses TNF-alpha-induced MMP-9 and cell invasion through inhibition of AP-1 activity in MDA-MB-231 human breast cancer cells

Abstract

Invasion of cancer cell induced by matrix metalloproteinase-9 (MMP-9) is one of pivotal steps in cancer metastasis. Herein, we investigated how cell invasion was regulated by berberine (BBR), an isoquinoline derivative alkaloid compound, in MDA-MB-231 human breast cancer cells. The basal level of MMP-9 activity and expression was dose-dependently increased by TNF-alpha, while TNF-a-induced MMP-9 gelatinase activity and expression was decreased by BBR. To investigate regulatory mechanism of TNF-alpha-induced MMP-9 expression, we pretreated cells with UO126 (MEK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor), respectively. Interestingly, TNF-alpha-induced MMP-9 activity and expression was decreased by UO126 and SB203580, but not by SP600125. Therefore, we further examined the effects of BBR on TNF-alpha-induced AP-1 DNA binding activity which is a downstream target of ERK and p38. Our data showed that TNF-alpha-induced AP-1 DNA binding activity was inhibited by BBR. Finally, we investigated the effect of BBR on TNF-alpha-induced cell invasion. TNF-alpha-induced cell invasion was significantly decreased by BBR treatment. Taken together, we suggest that TNF-alpha-induced MMP-9 expression and cell invasion are decreased by BBR through the suppression of AP-1 DNA binding activity in MDA-MB-231 human breast cancer cells.

Figure 1

The viability of MDA-MB-231 cells with treatment of berberine in different concentration. After serum-starvation for 16 h, cells treated with berberine at the indicated concentration for 48 h in fresh serum-free media. (A) Berberine structure. (B) Cell viabilities were analyzed by MTT assay as described in Materials and Methods. These results were representative of three independent experiments. Values shown are means ± SEM. Con; control.

Figure 2

TNF-α-induced MMP-9 activity and expression are dose-dependently decreased by berberine in MDA-MB-231 cells. (A) After serum-starvation for 16 h, cells were treated with TNF-α (20 ng/ml) for 24 h. (B) After serum-starvation for 16 h, cells were pretreated with berberine at the indicated concentration for 1 h and then treated with TNF-α (20 ng/ml) for 24 h. MMP-9 activity and expression were analyzed by Zymography (upper band) and Western blotting (lower band), respectively. These results were representative of three independent experiments. Values shown are means ± SEM. * P < 0.05, **P < 0.01 vs. control, † P < 0.05 vs. TNF-α-treated cells. Con; control.

Figure 3

TNF-α-induced MMP-9 activity and expression was inhibited by MEK and p38 inhibitors and TNF-α-induced AP-1 DNA binding activity were suppressed by berberine in MDA-MB-231 cells. (A) After serum-starvation for 16 h, cells were pretreated with 10 μM UO126, SB203580 and SP600126, respectively, for 30 min and then treated with 20 ng/mL TNF-α for 24 h. MMP-9 activity and expression were analyzed by Zymography (upper band) and Western blotting (lower band), respectively. (B) After serum-starvation for 16 h, cells were pretreated with 10 μM berberine for 1 h and then treated with 20 ng/mL TNF-α for the indicated times. (B) Using the nucleus protein, AP-1 DNA binding activity was analyzed by EMSA as described in Materials and Methods. These results were representative of three independent experiments. Values shown are means ± SEM. * P < 0.01 vs. control. † P < 0.01 vs. TNF-α-treated cells.Con; control, U; UO126, SB; SB203580, SP; SP600126.

Figure 4

TNF-α-induced cell invasion was suppressed by berberine in MDA-MB-231 cells. After seeding of 1×10⁵ cells/well, cells pretreated with 10 μM berberine for 1 h and then treated with 20 ng/mL TNF-α for 24 h. After 24 h incubation, cells on the bottom side of filter were fixed, stained and conunted. (A) Control; (B) TNF-α alone; (C) TNF-α with berberine. These results were representative of three independent experiments. Values shown are means ± SEM. * P < 0.01 vs. control, † P < 0.01 vs. TNF-α-treated cells. Con; control.