Exploring the Mechanism of Flaccidoxide-13-Acetate in Suppressing Cell Metastasis of Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the most common liver or hepatic cancer, accounting for 80% of all cases. The majority of this cancer mortality is due to metastases, rather than orthotopic tumors. Therefore, the inhibition of tumor metastasis is widely recognized as the key strategy for successful intervention. A cembrane-type diterpene, flaccidoxide-13-acetate, isolated from marine soft coral Sinularia gibberosa, has been reported to have inhibitory effects against RT4 and T24 human bladder cancer invasion and cell migration. In this study, we investigated its suppression effects on tumor growth and metastasis of human HCC, conducting Boyden chamber and Transwell assays using HA22T and HepG2 human HCC cell lines to evaluate invasion and cell migration. We utilized gelatin zymography to determine the enzyme activities of matrix metalloproteinases MMP-2 and MMP-9. We also analyzed the expression levels of MMP-2 and MMP-9. Additionally, assays of tissue inhibitors of metalloproteinase-1/2 (TIMP-1/2), the focal adhesion kinase (FAK)/phosphatidylinositide-3 kinases (PI3K)/Akt/mammalian target of the rapamycin (mTOR) signaling pathway, and the epithelial-mesenchymal transition (EMT) process were performed. We observed that flaccidoxide-13-acetate could potentially inhibit HCC cell migration and invasion. We postulated that, by inhibiting the FAK/PI3K/Akt/mTOR signaling pathway, MMP-2 and MMP-9 expressions were suppressed, resulting in HCC cell metastasis. Flaccidoxide-13-acetate was found to inhibit EMT in HA22T and HepG2 HCC cells. Our study results suggested the potential of flaccidoxide-13-acetate as a chemotherapeutic candidate; however, its clinical application for the management of HCC in humans requires further research.

Keywords: epithelial-mesenchymal transition; flaccidoxide-13-acetate; hepatocellular carcinoma; invasion; migration.

Figure 1

Cell viabilities of HA22T and HepG2 cells treated with flaccidoxide-13-acetate and a control (Mock: DMSO as the vehicle) after 24 h. (A) Cytotoxic effects on HA22T and HepG2 cells treated with flaccidoxide-13-acetate exhibiting a dose-dependent manner (# p < 0.05 and * p < 0.01). (B) Morphologies of HA22T and HepG2 cells after 24 h of incubation with 0–8-μM flaccidoxide-13-acetate. Scale bar = 20 μm. The results were obtained from three individual experiments.

Figure 2

Effects of the flaccidoxide-13-acetate treatment on HA22T and HepG2 cell migrations after treatment for 24 h. (A) Images of the migrations of flaccidoxide-13-acetate-treated HA22T and HepG2 cells as compared with controls (Mock: DMSO as the vehicle). Each image was representative of three individual experiments. (B) Inhibition ratios of flaccidoxide-13-acetate-treated HA22T and HepG2 cells as compared with controls (* p < 0.01). Data were calculated from three individual experiments. Scale bar = 20 μm.

Figure 3

Effects of 24-h flaccidoxide-13-acetate treatments on HA22T and HepG2 cell invasions. (A) Images of the invasions of flaccidoxide-13-acetate-treated HA22T and HepG2 cells as compared with the control (Mock: DMSO as the vehicle control). Each image was representative of three individual experiments. (B) Inhibition ratios of flaccidoxide-13-acetate-treated HA22T and HepG2 cells as compared with the controls (# p < 0.05 and * p < 0.01). Data were calculated from three individual experiments. Scale bar = 20 μm.

Figure 4

Activities of MMP-2/-9 and uPA and the protein levels of MMP-2, MMP-9, MMP-13, uPA, TIMP-1, and TIMP-2 after treatments of the HA22T and HepG2 cells with flaccidoxide-13-acetate at different concentrations for 24 h. (A) Images of the gelatin zymography of MMP-2/-9 and uPA activities. (B) Expression levels of MMP-2, MMP-9, MMP-13, uPA, TIMP-1, and TIMP-2. Mock: DMSO as the vehicle control.

Figure 5

Effects of flaccidoxide-13-acetate on the FAK/PI3K/Akt/mTOR signaling pathway in HA22T and Hep G2 cells. Protein expression levels of FAK, PI3K, Akt, and mTOR and the phosphorylation of PI3K, Akt, and mTOR after treatments with flaccidoxide-13-acetate for 24 h. β-actin: loading control. Mock: DMSO as the vehicle control.

Figure 6

Effects of flaccidoxide-13-acetate at different concentrations on the epithelial-to-mesenchymal transition (EMT) in HA22T and HepG2 cells. (A) Protein expression levels of β-catenin, N-cadherin, E-cadherin, and vimentin in cytosol. β-actin: loading control. Mock: DMSO as the vehicle control. (B) Protein expression levels of Snail in the nucleus. Internal controls: β-actin for cytosol and lamin A2 for the nucleus.

Figure 7

Hypothetical illustration of the flaccidoxide-13-acetate-associated pathway in HA22T and HepG2 human HCC cells.