Selaginellatamariscina attenuates metastasis via Akt pathways in oral cancer cells

Abstract

Background: Crude extracts of Selaginellatamariscina, an oriental medicinal herb, have been evidenced to treat several human diseases. This study investigated the mechanisms by which Selaginellatamariscina inhibits the invasiveness of human oral squamous-cell carcinoma (OSCC) HSC-3 cells.

Methodology/principal findings: Herein, we demonstrate that Selaginellatamariscina attenuated HSC-3 cell migration and invasion in a dose-dependent manner. The anti-metastatic activities of Selaginellatamariscina occurred at least partially because of the down-regulation of matrix metalloproteinases (MMP)-2 and MMP-9 gelatinase activity and the down-regulation of protein expression. The expression and function of both MMP-2 and MMP-9 were regulated by Selaginellatamariscina at a transcriptional level, as shown by quantitative real-time PCR and reporter assays. Chromatin immunoprecipitation (ChIP) data further indicated that binding of the cAMP response element-binding (CREB) protein and activating protein-1 (AP-1) to the MMP-2 promoter diminished at the highest dosage level of Selaginellatamariscina. The DNA-binding activity of specificity protein 1 (SP-1) to the MMP-9 promoter was also suppressed at the same concentration. Selaginellatamariscina did not affect the mitogen-activated protein kinase signaling pathway, but did inhibit the effects of gelatinase by reducing the activation of serine-threonine kinase Akt.

Conclusions: These results demonstrate that Selaginellatamariscina may be a potent adjuvant therapeutic agent in the prevention of oral cancer.

Figure 1. Effect of Selaginella tamariscina on cell viability and in vitro wound closure in HSC-3 cells.

(A) HSC-3 cells were treated with STE (0, 25, 50, 75 and 100 µg/mL) for 24 h before being subjected to a MTT assay for cell viability. The values represented the means ± SD of at least three independent experiments. (B) HSC-3 cells were wounded and then treated with vehicle (DMSO) or STE (0, 25, 50, 75 and 100 µg/mL) for 0h, 12h and 24 h in 10% FBS-containing medium. At 0, 12 and 24 h, phase-contrast pictures of the wounds at three different locations were taken.

Figure 2. Effect of STE on cell migration and invasion in HSC-3 cells.

(A) The cell migration and (B) cell invasion were measured using a Boyden chamber for 16h and 24 h with polycarbonate filters respectively. The migration and invasion abilities of HSC-3 cells were quantified by counting the number of cells that invaded to the underside of the porous polycarbonate as described in the Materials and Methods section. The values represented the means ± SD of at least three independent experiments. *p < 0.05 as compared with the vehicle group.

Figure 3. Effects of STE on the activity and protein level of MMP-2, MMP-9 and the protein level of the endogenous inhibitor TIMP-2 and TIMP-1.

(A and B) HSC-3 cells were treated with STE (0-100 µg/mL) for 24 h and then subjected to gelatin zymography to analyze the activity of MMP-2 and MMP-9, respectively. (C) HSC-3 cells were treated with STE (0-100 µg/mL) for 24 h and then subjected to western blotting to analyze the protein levels of MMP-2, MMP-9, TIMP-1 and TIMP-2. (D) Quantitative results of MMP-2, MMP-9, TIMP-1 and TIMP-2 protein levels which were adjusted with β-actin protein level. The values represented the means ± SD of at least three independent experiments. *p < 0.05 as compared with the vehicle group.

Figure 4. STE suppresses MMP-2 and MMP-9 expression at a transcriptional level.

Figure 5. Critical role of transcription factor in STE-induced transcriptional inhibition of MMP-2 and MMP-9 in HSC-3 cells.

Figure 6. Effects of STE on the MAPKs pathway and Akt signalings.