Anticancer effect of fufang yiliu yin on human hepatocellular carcinoma SMMC-7721 cells

Abstract

Chinese herbal medicine utilizes clinically effective adjuvants that can potentiate the effects of hepatectomy and molecule-targeted drugs for the treatment of hepatocellular carcinoma (HCC). The aim of this study was to investigate the possible molecular mechanisms underlying the antitumor effect of fufang yiliu yin (FYY) on HCC cells. We investigated the effects of FYY on the proliferation, migration, invasion, and apoptosis of SMMC-7721 cells in vitro and in mouse subcutaneous xenograft models in vivo. FYY significantly inhibited the proliferation of SMMC-7721 cells compared to that of normal hepatocytes; cell proliferation was blocked at the G₂/M phase in accordance with reduced expression of proliferating cell nuclear antigen. FYY treatment resulted in the activation of caspase-8, caspase-3 and poly (ADP-ribose) polymerase, with reduced protein levels of tumor necrosis factor receptor-associated factor 2, indicating an induction of cell apoptosis. In addition, we observed decreases in the protein expression of matrix metalloproteinase-2 and -9 along with an inhibition of cell migration and invasion after FYY treatment. Furthermore, FYY treatment significantly inhibited the growth of tumors in vivo. These data demonstrate the strong inhibitory effects of FYY on SMMC-7721 cells, and we propose FYY as a novel potential anticancer adjuvant.

Keywords: Chinese herbal medicine; Hepatocellular carcinoma; anticancer.

Figure 1

FYY inhibits the proliferation of SMMC-7721 cells. A. FYY inhibited the proliferation of SMMC-7721 cells, but not normal HL-7702 hepatocyte cells, treated for 24, 48, and 72 h in a dose- and time- dependent manner. Cell density and morphology changes are shown (× 100 magnification). B. The colony formation ability was decreased after treatment with FYY (× 100 magnification). C. Representative Western blot and quantitation of PCNA expression showing a dose-dependent decrease with FYY treatment. D. Cell-cycle progression was arrested at the G₂/M phase by FYY. Data are expressed as the means ± SDs from three separate experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. PBS.

Figure 2

FYY induces apoptosis of SMMC-7721 cells. Hoechst staining (A) and flow cytometric analysis (B) indicate that FYY induced cellular apoptosis in a dose-dependent manner. (C) Expression levels of apoptosis-related proteins (TRAF2, caspase-8, caspase-3, and PARP) were altered by FYY treatment as determined by Western blotting. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. PBS.

Figure 3

FYY inhibits SMMC-7721 cell migration and invasion. Wound healing (A) and Transwell invasion (B) assays indicate that FYY inhibited SMMC-7721 cell migration. (C) FYY also inhibited the invasion of SMMC-7721 cells into Matrigel. (D) Expression levels of extracellular matrix degradation-related proteins MMP-2 and MMP-9 were dose-dependently decreased with FYY treatment as determined by Western blotting. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. PBS.

Figure 4

FYY inhibits the growth of SMMC-7721 cells in vivo. (A) Subcutaneous xenograft tumors after 33 d. Animals treated with FYY had tumors with significantly smaller volumes (B) and lower weights (C). *P < 0.05 and **P < 0.01 vs. control (saline-treated animals).