Inhibitory effects of Ganoderma lucidum triterpenoid on the growth and metastasis of hepatocellular carcinoma

Abstract

Objective: To investigate the inhibitory effects and mechanisms of triterpenoids from Ganoderma lucidum (G. lucidum triterpenoids) on the growth and metastasis of hepatocellular carcinoma (HCC) both in vitro and in vivo.

Methods: In in-vitro experiments, the inhibitory effects of G. lucidum triterpenoids on human HCC SMMC-7721 cell lines were investigated by observing the proliferation, apoptosis, migration and invasion phenotypes of the cell line and assessing the cell cycles as well as the cell apoptosis and proliferation. In in-vivo experiments, nude mouse SMMC-7721 tumor models were established and divided into control group, treatment group A (low concentration group) and treatment group B (high concentration group) according to the treatment models received. Magnetic resonance imaging (MRI) was performed 3 times on each mouse model to calculate their tumor volumes. The liver and kidney functions of the models were evaluated. Tissues harvested from their solid organs were subjected to HE staining, and the tumor tissues were subjected to HE staining and immunohistochemical staining (E-cad, Ki-67, and Tunel), respectively.

Results: i. In in-vitro experiments, G. lucidum triterpenoids could inhibit the growth of human HCC SMMC-7721 cell lines via regulating their proliferation and apoptosis phenotype. ii. In in-vivo experiments, the comparison of tumor volumes of mouse models obtained from the second and third MIR scanning was found to be statistically significant between the control group and treatment group A (P<0.05); and statistically significant differences were also found in the tumor volumes from the second and third MRI scanning between the control group and treatment group B (P<0.05). iii. No significant acute injuries or adverse effects were observed in the liver or kidney of the nude mice.

Conclusion: G. lucidum triterpenoids could inhibit the growth of tumor cells via blocking their proliferation, accelerating apoptosis, and inhibiting migration and invasion, without marked toxic effects on normal organs and tissues in the body.

Keywords: G. lucidum triterpenoids; Hepatocellular carcinoma; MRI; nude mouse model; pathology.

Figure 1

Experimental results. A. Effects of different concentrations of G. lucidum triterpenoids on the activity and cell cycles of HCC cells detected by CCK-8 at different time points (***P<0.001, vs. 0 ug/mL, ###P<0.001, vs. 2.5 ug/mL, &&&P<0.001, vs. 5 ug/mL). B. Bar charts showing the effects of different concentrations of G. lucidum triterpenoids on HCC cell cycles detected by flow cytometry (**P<0.01, ***P<0.001, vs. Control, ###P<0.001, vs. 2.5 ug/mL, &P<0.05, vs. 5 ug/mL). C. Bar charts and the flow cytometry figure showing the effects of different concentrations of G. lucidum triterpenoids on HCC apoptosis (***P<0.001, vs. Control, ###P<0.001, vs. 2.5 ug/mL, &&&P<0.001, vs. 5 ug/mL). One-way ANOVA was used to detect the differences for further analysis and Turkey method was used for between-group comparison.

Figure 2

Effects of different concentrations of G. lucidum triterpenoids on HCC cell apoptosis, metastasis and invasion. A. Effects of different concentrations of G. lucidum triterpenoids on HCC cell apoptosis, as shown in the TUNEL fluorescent images and bar charts (***P<0.001, vs. 0 ug/mL, ##P<0.01, ###P<0.001, vs. 2.5 ug/mL, &&&P<0.001, vs. 5 ug/mL). B. The migration ability of HCC cells after the treatment of different concentrations of G. lucidum triterpenoids by wound healing assay (***P<0.001, vs. Control, #P<0.05, ##P<0.01, vs. 2.5 ug/mL). C. The invasion ability of HCC cells after the treatment of different concentrations of G. lucidum triterpenoids by Transwell assay (**P<0.01, ***P<0.001, vs. Control, ###P<0.001, vs. 2.5 ug/mL, &&&P<0.001, vs. 5 ug/mL). One-way ANOVA was used to detect the differences for further analysis and Turkey method was used for between-group comparison. All figures were zoomed in 200 times.

Figure 3

Comparisons of the tumor volume and immunohistochemical staining results of each organ and tumor tissue. A. Three MRI results of the tumor tissues of nude mice in each group. B. The average tumor volumes of nude mice in each group after three MRI scans. C. HE staining images of the heart, liver, spleen, lung, and kidney tissues of nude mice in each group. D. HE staining images of the tumor tissues of nude mice in each group. E. E-cad, Ki-67 and Tunel staining images of tumor sections of nude mice in each group. Note: All figures were zoomed in 400 times. n=6 in all three groups.