Sinomenine influences capacity for invasion and migration in activated human monocytic THP-1 cells by inhibiting the expression of MMP-2, MMP-9, and CD147

Abstract

Aim: The aim of this study was to investigate the mechanism of the effects of Sinomenine (SIN) on the invasion and migration ability of activated human monocytic THP-1 cells (A-THP-1). Sinomenine is a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum.

Methods: Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA). Cells were treated with different concentrations of SIN. The invasion and migration ability of cells was tested by in vitro transwell assays. The levels of CD147 and MMPs were evaluated by flow cytometric analysis and zymographic analysis, respectively. The mRNA expression of CD147, MMP-2, and MMP-9 was measured by RT-PCR.

Results: The invasion and migration ability of A-THP-1 cells was significantly inhibited by SIN in a concentration-dependent fashion; at the same time, the levels of CD147, MMP-2, and MMP-9 were markedly down-regulated. This inhibitory effect was most notable at concentrations of 0.25 mmol/L and 1.00 mmol/L (P<0.01).

Conclusion: A possible mechanism of the inhibitory effect of SIN on cell invasion and migration ability is repression of the expression of MMP-2 and MMP-9, which strongly correlates with the inhibition of CD147 activity.

Figure 1

Growth curve of A-THP-1 cells in RPMI 1640 with 10% FCS. Cells (5×10⁸ cells/L) were incubated with various concentrations of SIN (0, 0.01, 0.05, 0.25, and 1.00 mmol/L) for 48 h. The intensity ratio (%) indicates the cell viability at other drug concentrations as compared with the control. n =6. Mean±SD. ^aP>0.05 vs control.

Figure 2

Cell invasion and migration assay in SIN (0, 0.01, 0.05, 0.25, and 1.00 mmol/L) treated A-THP-1 cells (5×10⁸ cells/L). Mean cell counts from 6 random fields and data represent the mean±SD of three independent experiments. ^aP>0.05, ^bP<0.05, ^cP<0.01 vs control.

Figure 3

SIN repressed the mRNA expression of CD147, MMP-2, and MMP-9 as tested by RT-PCR. (A) A-THP-1 cells (5×10⁸ cells/L) were treated with various concentrations of SIN for 48 h. The data are representative of three independent experiments. (B) Quantification of RT-PCR data. Values correspond to the mean±SD of three independent experiments. ^aP>0.05, ^bP<0.05, ^cP<0.01 vs control.

Figure 4

(A) Flow cytometric analysis of the effect of SIN on the expression of CD147 in A-THP-1 cells. A-THP-1 cells (5×10⁸ cells/L) were incubated with SIN for 48 h. Expression of CD147 was analyzed with a Cytoron flow cytometer. (B) Quantification of flow cytometric analysis data. Values correspond to the mean±SD of three independent experiments. ^aP>0.05, ^bP<0.05, ^cP<0.01 vs control.

Figure 5

Gelatin zymography for the determination of MMP-2 and MMP-9 activities in SIN treated A-THP-1 cells. (A) A-THP-1 cells (5×10⁸ cells/L) were treated with various concentrations of SIN (0, 0.01, 0.05, 0.25, and 1.00 mmol/L) for 48 h, and activities of MMP-2 and MMP-9 in conditioned media were evaluated by electrophoresis of soluble protein on a gelatin containing 7.5% polyacrylamide gel. (B) Areas and relative intensities of gelatin digested bands by MMP-2 and MMP-9 were quantified by densitometry and expressed as relative activity compared to that of the RPMI-1640 control group. Values correspond to the mean±SD of three independent experiments. ^aP>0.05, ^bP<0.05, ^cP<0.01 vs control.