Reduction of COX-2 through modulating miR-124/SPHK1 axis contributes to the antimetastatic effect of alpinumisoflavone in melanoma

Abstract

Alpinumisoflavone (AIF) is a naturally occurring flavonoid that is a major bioactive component of the medicinal plant Derris eriocarpa. In this study we evaluated the antimetastatic effect of AIF and investigated the underlying mechanism of action using in vitro and in vivo models of melanoma. We found that AIF impaired the metastatic potential of A375 and SK-MEL-1 human melanoma cells by promoting cell differentiation as assessed by melanin content, protoporphyrin IX accumulation, and tissue transglutaminase activity. In addition, AIF inhibited cell adhesion, migration, and invasion in melanoma cells. We found that AIF treatment decreased cyclooxygenase-2 (COX-2) expression, and COX-2 overexpression attenuated the inhibitory effects of AIF on the metastatic behaviors of melanoma cells. AIF dose-dependently increased microRNA-124 (miR-124) levels and decreased levels of sphingosine kinase 1 (SPHK1), a target of miR-124. In a mouse model of melanoma, AIF suppressed lung metastasis. Taken together, our findings suggest that AIF inhibits metastasis in melanoma by modulating COX-2 expression, at least in part, through targeting the miR-124/SphK1 axis. Our study provides evidence that AIF may be useful as an antimetastatic agent in the treatment of melanoma.

Keywords: Alpinumisoflavone; COX-2; SPHK1; melanoma; metastasis; miR-124.

Figure 1

AIF reduces cell viability and promotes differentiation in A375 and SK-MEL-1 human melanoma cells. A. AIF treatment led to a time- and dose-dependent loss of cell viability. To determine the effect of AIF on cell differentiation, cells were treated with AIF at the indicated concentrations for 48 hours. B. AIF treatment increased the melanin content of cells. C. AIF treatment increased PpIX accumulation. D. AIF enhanced TG2 activity. *P < 0.05, **P < 0.01.

Figure 2

AIF suppresses migration and invasion in A375 and SK-MEL-1 human melanoma cells. To demonstrate the effect of AIF on migration and invasion, cells were incubated with AIF at the indicated concentrations. A. Treatment with 5 or 10 μM AIF for 24 hours decreased cell migration. B. Treatment with 5 or 10 μM AIF for 48 hours suppressed cell invasion. *P < 0.05, **P < 0.01.

Figure 3

AIF promotes cell differentiation by suppressing COX-2 expression. Cells were incubated with AIF at the indicated concentrations for 24 hours. A. AIF treatment dose-dependently decreased COX-2 mRNA levels. B. AIF treatment dose-dependently decreased COX-2 protein levels. C. Transfection of a COX-2 overexpression plasmid attenuated the effect of AIF on melanin content. D. COX-2 overexpression attenuated AIF-induced PpIX accumulation. E. COX-2 overexpression attenuated the effect of AIF on TG2 activity. **P < 0.01 vs. control, ^^P < 0.01 vs. AIF.

Figure 4

The anti-invasive effect of AIF in melanoma cells is mediated by downregulating COX-2 expression. Melanoma cells were incubated with AIF at the indicated concentrations for 24 hours. A. COX-2 overexpression attenuated the inhibitory effect of AIF on cell migration. B. COX-2 overexpression attenuated the inhibitory effect of AIF on cell invasion. **P < 0.01 vs. control, ^^P < 0.01 vs. AIF.

Figure 5

AIF represses COX-2 expression by modulating miR-124/SPHK1 signaling. Cells were pretreated for 24 hours with AIF (10 μM unless otherwise noted). A. AIF treatment dose-dependently decreased SPHK1 protein levels in melanoma cells. B. AIF treatment dose-dependently increased mIR-124 expression in melanoma cells. C. MiR-124 knockdown significantly attenuated the suppressive effects of AIF on SPHK1 expression, as demonstrated by western blot analysis. D. Overexpression of miR-124 significantly decreased SPHK1 transcription, as assessed by the luciferase reporter assay. E. Both MiR-124 knockdown and SPHK1 overexpression significantly attenuated the suppressive effect of AIF on COX-2 mRNA expression. F. Both MiR-124 knockdown and SPHK1 overexpression significantly attenuated the suppressive effect of AIF on COX-2 protein expression. **P < 0.01 vs. control, ^^P < 0.01 vs. AIF.

Figure 6

MiR-124/SPHK1 signaling is involved in the antimetastatic effect of AIF. Cells were pretreated for 24 hours with AIF (10 μM if not otherwise noted). A. COX-2 overexpression attenuated the effect of AIF on melanin content in melanoma cells. B. Both MiR-124 knockdown and SPHK1 overexpression significantly attenuated the effect of AIF on PpIX accumulation. C. Both MiR-124 knockdown and SPHK1 overexpression significantly attenuated the effect of AIF on TG2 activity. D. Both MiR-124 knockdown and SPHK1 overexpression significantly attenuated AIF-mediated inhibition of cell migration. E. Both MiR-124 knockdown and SPHK1 overexpression significantly attenuated AIF-mediated inhibition of cell invasion. **P < 0.01.

Figure 7

AIF suppresses B16-F10 murine melanoma cell lung metastasis in vivo. Xenograft-bearing mice were treated for 24 days with vehicle or AIF (20 or 50 mg/kg/day) by intragastric administration. Representative images of lung metastasis and the number of metastatic nodules in the lungs are shown. A. AIF significantly suppressed lung metastasis in vivo. B. AIF increased miR-124 expression in vivo. C. AIF treatment decreased expression levels of COX-2 and SPHK1 in vivo. *P < 0.05 (n = 8).