Anti-tumor effect of volatile oil from Houttuynia cordata Thunb. on HepG2 cells and HepG2 tumor-bearing mice

Abstract

The aim of this paper is to study the anti-tumor mechanism of volatile oil from Houttuynia cordata Thunb. (sodium new houttuyfonate, SNH). In vitro, SNH exhibited a concentration-dependent cytotoxic effect against four human cancer lines (HepG2, A2780, MCF-7, SKOV-3). SNH treatment with different concentrations induced HepG2 cells to exhibit varying degrees of morphological changes in apoptotic features, such as round shape, cell shrinkage and formation of apoptotic body. It was observed that SNH caused the decrease in Bcl-2 mRNA expression and triggered the apoptosis of HepG2 cells. Wound healing assay and RT-PCR results showed that the decrease in the expression level of MMP9 and VEGF was observed in HepG2 cells after treatment with SNH for 48 h, suggesting that the extracellular matrix pathway degradation was involved in the HepG2 cells migration. Moreover, we got an insight into the binding mode of SNH into the MMP9 active site through 3D pharmacophore models. Docking study and molecular dynamics (MD) simulation analysis sheds light on that SNH was completely embedded into the MMP9 active site and formed hydrogen bonds with key catalytic residues of MMP9, including Ala191, His190, Ala189 and Glu227. The prediction of SNH binding interaction energies in the MMP9 was almost in good agreement with the original inhibitor EN140. In vivo experiments, both SNH and cyclophosphamide significantly reduced tumor weights and their tumor inhibitory rates were 50.78% and 82.61% respectively. This study demonstrated that SNH was an apoptosis inducer in HepG2 cells. SNH has four possible functions, that it could induce apoptosis by mitochondria pathway in HepG2 cells, inhibit the tumor growth, regulate Bcl-2 family mRNA expression and effectively subdue migration of hepatocellular carcinoma cells by decreasing the expression of MMP9 and VEGF. Therefore, SNH might be a potential candidate drug for the treatment of hepatocellular carcinoma, which could provide a reference for further clinical research.

Fig. 1. Cytotoxic effect of SNH against four different human tumor cell lines after 48 h treatment at 37 °C. Cell viability percentage of hepatoma HepG2 cell line (black), breast cancer MCF-7 cell line (green), ovarian cancer SKOV-3 cell line (pink) and ovarian cancer A2780 cell line (blue) can be observed. Dada are shown as mean ± SD of 3 parallel wells. Statistically significant differences were found between the cytotoxic effects exhibited by the SNH in the HepG2 cell line at 200 μg mL⁻¹ (*) and 300, 400, 500 μg mL⁻¹ (**). (Independent-samples t test, *P < 0.05, **P < 0.01 versus the control group).

Fig. 2. SNH induces apoptosis in HepG2 cells. HepG2 cells were treated with SNH (0, 200, 300, 400 and 500 μg mL⁻¹) for 48 h. (A) Morphological changes of HepG2 cells were observed with a phase contrast microscope (200× magnification). (B) The nuclear morphology changes of HepG2 cells were detected by Hoechst 33258 staining (100× magnification).

Fig. 3. HepG2 cells were treated with different concentrations of SNH (0, 100, 200, 300, 400 and 500 μg mL⁻¹) for 48 h, and DNA fragments was collected and 20 μL of final sample was loaded in all lanes. Electrophoresis was performed on 1.2% agarose gel. Data shown are representative of three separate experiments.

Fig. 4. HepG2 cells were treated with SNH (0, 100, 200, 300, 400 and 500 μg mL⁻¹) for 48 h, and apoptosis was measured by flow cytometry after staining with annexin V/PI, (independent-samples t test, **P < 0.01, ***P < 0.001 versus the control group).

Fig. 5. SNH inhibited HepG2 cells migration in wound healing. HepG2 cells were treated with various concentrations of SNH (0, 100, 200, 300, and 400 μg mL⁻¹) for 48 h. (A) Representative images (100× magnification) show cells migrating into wounded area in an in vitro scratch wound healing assay. (B) Quantification of the endothelial wound repair. The distance of cell migration to the wound area was measured in HepG2 cells after wounding. 0 μg mL⁻¹ treatment was used as the standardized control for quantification. Values are means ± SD from three independent experiments. (Independent-samples t test, *P < 0.05; **P < 0.01; ***P < 0.001 versus the control group).

Fig. 6. Schematic representation of the crystal structures of the MMP9 catalytic domain bound to inhibitors SNH ligand and EN140 ligand. (A) Stereo diagrams of the MMP9 active site. Superposition of the SNH ligand and EN140 ligand active sites. The SNH ligand structure is coloured blue; the EN140 ligand structure is coloured green. (B) Total interaction energy statistics of SNH ligand and EN140 ligand complex surface on the MMP9. (C) A representation of the interaction bonds between the MMP9 catalytic domain and the EN140 ligand inhibitor. These results were analyzed and visualized using PyMOL (http://www.pymol.org). (D) Close-up of the MMP9 and SNH ligand complex. Four short hydrogen bonds are formed between Ala189, His190, Ala19, Glu227 and SNH ligand. Attractive charge is orange, conventional hydrogen bond is green, carbon hydrogen bond is light green and hydrophobic bond is shown in pink.

Fig. 7. Effect of SNH on the expression of apoptosis and migration-related mRNA in HepG2 cells assayed RT-PCR analysis. GADPH was employed as loading control, (independent-samples t test, *P < 0.05; **P < 0.01; ***P < 0.001 versus the control group).

Fig. 8. Effect of SNH on tumors from HepG2 bearing mice. (A) Showed tumor tissues of each group. (B) Displayed mean tumor weights and tumor inhibitory rates. Data were presented as mean ± SD. (Independent-samples t test, **P < 0.01 versus the PBS group).