Herbal compound "Songyou Yin" attenuates hepatoma cell invasiveness and metastasis through downregulation of cytokines secreted by activated hepatic stellate cells

Abstract

Background: Activated hepatic stellate cells (aHSCs) play an important role in the progression of hepatocellular carcinoma (HCC). Here, we determined if cytokines secreted in response to the herbal compound "Songyou Yin" (SYY) treatment of aHSCs could influence invasiveness and metastatic capabilities of hepatoma cells.

Methods: Primary rat hepatic stellate cells (HSCs) were isolated, activated, divided into SYY treated and untreated (nSYY) groups, and conditioned media (CM-SYY and CM-nSYY, respectively) were collected. The hepatoma cell line, McA-RH7777 was cultured for 4 weeks with SYY, CM-SYY, and CM-nSYY, designated McA-SYY, McA-SYYCM and McA-nSYYCM. The invasiveness and metastatic capabilities were evaluated using Matrigel invasion assay in vitro and pulmonary metastasis in vivo. Matrix metalloproteinase-2 (MMP-2), MMP-9, E-cadherin, N-cadherin, and vimentin protein levels in McA-SYYCM and McA-nSYYCM were evaluated by Western blot. Cytokine levels in conditioned media were tested using enzyme-linked immunosorbent assay (ELISA).

Results: Matrigel invasion assay indicated that the number of McA-SYYCM cells passing through the basement membrane was less than in McA-nSYYCM cells (P < 0.01). Similar results were also observed in vivo for lung metastasis. McA-SYYCM cells showed less pulmonary metastasis capabilities than McA-nSYYCM cells (P < 0.001). The reduced expression of MMP-2 and reversed epithelial to mesenchymal transition with E-cadherin upregulation, and N-cadherin and vimentin downregulation were also found in McA-SYYCM compared to McA-nSYYCM. Metastasis-promoting cytokines hepatocyte growth factor, interleukin-6, transforming growth factor-β1, and vascular endothelial growth factor were markedly decreased in CM-SYY compared to CM-nSYY.

Conclusions: SYY attenuates hepatoma cell invasiveness and metastasis capabilities through downregulating cytokines secreted by activated hepatic stellate cells.

Figure 1

Freshly isolated HSCs from buffalo rats were quiescent, rich in lipid droplets and with spontaneous green fluorescence. Fourteen days after isolation, HSCs were activated and developed into fibroblast-like cells (A). Activated HSCs were immunostained with desmin (B), and α-SMA (C) followed by FITC-conjugated secondary antibody, and nuclei were stained with DAPI.

Figure 2

The number of pulmonary nodules implanted with McA-SYYCM cells was less than the nodule number from McA-nSYYCM cells. McA-SYY cell implants had the smallest number of nodules (A, B). The aHSCs and McA-RH7777 cells, which were treated with 2 mg/ml SYY for 4 h, 8 h, and 12 h, demonstrated no increased release of LDH, indicating no acute cytotoxicity (C).

Figure 3

The transwell assays demonstrated that McA-SYYCM cells passed through the basement membrane in less number than McA-nSYYCM cells, and McA-SYY cells were the lowest in number (A). The number of invading cells is expressed as mean ± SD (B). Based upon western blots, E-cadherin was upregulated, while N-cadherin, vimentin, and MMP-2 were downregulated in McA-SYYCM cells compared to McA-nSYYCM cells. MMP-9 levels were low in both McA-SYYCM and McA-nSYYCM cells, and were not statistically different (C).