Inhibitory effects of tanshinone II-A on invasion and metastasis of human colon carcinoma cells

Abstract

Aim: To investigate the effects and possible mechanisms of tanshinone II-A, an alcohol extract of the root of Salvia miltiorrhiza Bunge, on tumor invasion and metastasis of human colon carcinoma (CRC) cells.

Methods: The effects of tanshinone II-A on invasion and metastasis of CRC cell lines HT29 and SW480 were evaluated by in vitro and in vivo assays. Western blotting was used to investigate possible molecular mechanisms of tanshinone II-A anti-cancer actions.

Results: Tanshinone II-A inhibited migration and invasion of CRC cells in a dose-dependent manner. The inhibitory effect also depended on time, with the most significant effects observed at 72 h. Tanshinone II-A also significantly inhibited in vivo metastasis of colon carcinoma SW480 cells. It inhibited in vitro and in vivo invasion and metastasis of CRC cells by reducing levels of urokinase plasminogen activator (uPA) and matrix metalloproteinases (MMP)-2 and MMP-9, and by increasing levels of tissue inhibitor of matrix metalloproteinase protein (TIMP)-1 and TIMP-2. Tanshinone II-A was also shown to suppress the nuclear factor-kappaB (NF-kappaB) signal.

Conclusion: Tanshinone II-A inhibited in vitro and in vivo invasion and metastasis of CRC cells. The effect resulted from changes in the levels of uPA, MMP-2, MMP-9, TIMP-1 and TIMP-2, and apparent inhibition of the NF-kappaB signal transduction pathway.

Figure 1

Migratory ability of SW480 cells. Cell migration was evaluated by wound healing assay after 24 h incubation with 0, 0.5, 1.0, or 2 mg/L tanshinone II-A.

Figure 2

Effects of tanshinone II-A on metastasis of SW480 cell in nude mice. (A) An in vivo metastasis model was made by injection of SW480 tumor tissue into the livers of nude mice. Experimental and control groups had 10 mice each. Groups were treated with tanshinone II-A at 0, 5, 20, or 80 mg·kg⁻¹·d⁻¹. Four weeks later, the mice were sacrificed and the number of visible tumors on the liver surface was counted. ^bP<0.05, ^cP<0.01 vs the control group without tanshinone II-A. (B) Serial sections of liver tissues were HE dyed and observed under a light microscope.

Figure 3

The effect of tanshinone II-A on uPA, MMP-2, MMP-9, TIMP-1, and TIMP-2 in colon carcinoma cells. A representative experiment of three with similar results is shown. The expression of uPA, MMP-2, MMP-9, TIMP-1 and TIMP-2 in the cytoplasm, and the active forms of MMP-2, and MMP-9 in the supernatant were evaluated by Western blot. β-actin was used as an internal control. (A) After 48 h of incubation with 0, 1.0, 1.5, or 2 mg/L of tanshinone II-A, the levels of uPA, MMP-2 and MMP-9 proteins in colon carcinoma cells decreased, and the levels of TIMP-1 and TIMP-2 proteins in colon carcinoma cells increased dose-dependently, compared to untreated cells (P<0.05). (B) After 24, 48, 72, or 96 h incubation with 2 mg/L tanshinone II-A, the levels of uPA, MMP-2 and MMP-9 proteins in colon carcinoma cells decreased, and the levels of TIMP-1 and TIMP-2 proteins in colon carcinoma cells increased, time-dependently (P<0.05). β-actin was used as an internal control. (C) The effect of tanshinone II-A on p-p65 in colon carcinoma cells. A representative experiment of three with similar results is shown. The expression of p-p65 in the nucleus was evaluated by Western blot. β-actin was used as an internal control.