THE HERBAL MIXTURE XIAO-CHAI-HU TANG (XCHT) INDUCES APOPTOSIS OF HUMAN HEPATOCELLULAR CARCINOMA HUH7 CELLS IN VITRO AND IN VIVO

Abstract

Background: Xiao-Chai-Hu Tang (XCHT) is an extract of seven herbs with anticancer properties, but its mechanism of action is unknown. In this study, we evaluated XCHT-treated hepatocellular carcinoma (HCC) for anti-proliferative and pro-apoptotic effects.

Materials and methods: Using a hepatic cancer xenograft model, we investigated the in vivo efficacy of XCHT against tumor growth by evaluating tumor volume and weight, as well as measuring apoptosis and cellular proliferation within the tumor. To study the effects of XCHT in vitro, we measured the cell viability of XCHT-treated Huh7 cells, as well as colony formation and apoptosis. To identify a potential mechanism of action, the gene and protein expression levels of Bax, Bcl-2, CDK4 and cyclin-D1 were measured in XCHT-treated Huh7 cells.

Results: We found that XCHT reduced tumor size and weight, as well as significantly decreased cell viability both in vivo and in vitro. XCHT suppressed the expression of the proliferation marker Ki-67 in HCC tissues and inhibited Huh7 colony formation. XCHT induced apoptosis in HCC tumor tissues and in Huh7 cells. Finally, XCHT altered the expression of Bax, Bcl-2, CDK4 and cyclin-D1, which halted cell proliferation and promoted apoptosis.

Conclusion: Our data suggest that XCHT enhances expression of pro-apoptotic pathways, resulting in potent anticancer activity.

Keywords: Xiao-Chai-Hu Tang; apoptosis; hepatocellular carcinoma; proliferation.

Figure 1

Effect of Xiao-Chai-Hu Tang (XCHT) on the viability and morphology of Huh7 cells. (A) Cell viability was determined by the MTT assay after Huh7 cells were treated with 0.5-1.5 mg/ml XCHT for 24, 48 or 72 h. The data were normalized to the viability of control cells (100%). Data are the averages with standard deviation (SD; error bars) from 3 independent experiments. The symbols (#), (*), and (&) indicate statistical significance compared to control cells (P<0.05), for each indicated timepoint. (B) The Huh7 cells were treated with the 0.5-1.5 mg/ml XCHT for 24 h, and morphological changes were observed using phase-contrast microscopy. The images were captured at a magnification of 200x. Images are representative of 3 independent experiments.

Figure 2

Effect of XCHT on tumor growth in hepatocellular carcinoma (HCC) xenograft mice. After tumor development, the mice were given intra-gastric administration of 14.2 g/kg of XCHT or PBS daily for 21 days. Tumor volume (A), tumor weight (B), and body weight (C) were measured. Data shown are averages with SD (error bars) from 10 mice in each group (n = 10). * P< 0.05, versus controls.

Figure 3

Effect of XCHT on cell proliferation in HCC xenograft mice and Huh7 cells. (A) Ki-67 assay in tumor tissues (400 χ). Data shown are averages with SD (error bars) from 6 individual mice in each group (n = 6). * P < 0.05, versus controls. (B) Huh7 cell colony formation assay. Data are averages with SD (error bars) from at least three independent experiments. *P < 0.01, versus control cells.

Figure 4

Effect of XCHT on apoptosis in both HCC xenograft mice and Huh7 cells. (A) TUNEL assay in tumor tissues (400 x). Data shown are averages with SD (error bars) from 6 individual mice in each group (n = 6). *P < 0.05, versus controls; (B) Huh7 cells were treated with 0.5-1.5 mg/ml XCHT for 24 h and stained with Hoechst 33258. Images were visualized using a phase-contrast fluorescence microscope. The images were captured at a magnification of 400x. Images are representative of 3 independent experiments.

Figure 5

Effect of XCHT on the expression of Bcl-2, Bax, cyclin-D1 and CDK4 in HCC xenograft mice. (A) Four tumors were randomly selected from each group, and the mRNA or protein expression levels of Bcl-2, Bax, cyclin-D1, and CDK4 were determined by RT-PCR and Western blot analysis. GAPDH or β-actin were used as the internal controls. Data shown are representative samples. (B) Densitometric analysis of gene and protein expression levels. The data were normalized to the mean mRNA or protein expression levels of untreated control mice (100%). The symbols (*), (&), (#), and (§) indicate statistical significance versus controls (P< 0.05), for Bax, Bcl-2, CDK4 or cyclin-D1.

Figure 6

Effect of XCHT on the expression of Bcl-2, Bax, cyclin-D1 and CDK4 in Huh7 cells. The cells were treated with 0.5-1.5 mg/ml XCHT for 24 h. (A) The mRNA and protein levels of Bcl-2, Bax, CDK4 and cyclin-D1 were