The Synergistic Antitumor Effect of Tanshinone IIA Plus Adriamycin on Human Hepatocellular Carcinoma Xenograft in BALB/C Nude Mice and Their Influences on Cytochrome P450 CYP3A4 In Vivo

Abstract

Objective: Hepatocellular carcinoma is one of the most common diseases that seriously threaten human life and health. In this study, we evaluated the inhibitory effect of tanshinone IIA (Tan IIA) combined with adriamycin (ADM) on human hepatocellular carcinoma and developed a platform to assess the function if Chinese herbal ingredients combined with chemotherapy drugs have synergistic antitumor effects in vivo.

Methods: Established animal model of human hepatocarcinoma HepG2 cell in nude mice. Mice were divided into model control group, Tan IIA group, ADM group, and Tan IIA + ADM group. The changes from general condition, weight, tumor volume, and inhibition rate were observed. The data were gathered from serum AST level and histopathological changes. The content and activity of cytochrome P450 were determined by spectrophotometric analysis. CYP3A4 protein expression was analyzed by western blotting. The binding model crystal structure of Tan IIA and ADM with pregnane X receptor (PXR) was evaluated by Discovery Studio 2.1.

Results: A combination of Tan IIA with ADM could improve life quality by relieving ADM toxicity, decreasing tumor volume, declining serum AST level, and improving liner pathological section in tumor-bearing mice. The inhibitory rates of Tan IIA, ADM, and cotreatment were 32.77%, 60.96%, and 73.18%, respectively. The Tan IIA group significantly enhanced the content of cytochrome b5, P450, and erythromycin-N-demethylase activity. CYP3A4 protein expression was enhanced obviously by the Tan IIA + ADM group. Virtual molecular docking showed that both Tan IIA and ADM could be stably docked with the same binding site of PXR but different interactions.

Conclusions: Tan IIA in combination with ADM could improve the life quality in tumor-bearing mice and enhance the antitumor effect. The Tan IIA group increased the concentration of cytochrome P450 enzymes and activity. Combined Tan IIA with ADM could upregulate the CYP3A4 protein expression and make relevant interaction with protein PXR by virtual docking.

Figure 1

Experimental design of the xenograft animal model. HepG2 cell tumor xenograft in BALB/c nude mice. Mice were divided randomly into four groups: model control group, Tan-IIA (15 mg/kg of body weight, day 1∼7, day 17∼23), ADM (4 mg/kg of body weight, day 1∼3, day 17∼19), and Tan IIA + ADM group (Tan-IIA 15 mg/kg plus ADM 4 mg/kg).

Figure 2

Changes of general condition and tumor growth in HepG2 tumor-bearing mice. (a) Tumor-bearing nude mice before treatments. (b) The changes of general condition and weight growth during treatments in tumor-bearing nude mice. (c) The photograph shows the representative mice tumors in the different treatment groups.

Figure 3

Effects of Tan IIA or AMD treatments on the HepG2 tumor-bearing mice. (a) Body weight in different treatment groups. (b) Tumor volume of mice was recorded during treatments. (c, d) Effects of different treatments on final tumor volume and weight when compared on the 23th day. N = 5 except for the Figures 3(c) and 3(d) in the ADM group (n = 4) and the Tan IIA + ADM group (n = 3) ^∗P < 0.01 versus control group; ^▲P < 0.01 versus Tan IIA group; ^#P < 0.01 versus ADM group.

Figure 4

Effect of Tan IIA and ADM on the serum AST level in HepG2 tumor-bearing nude mice (n = 5, ^∗P < 0.01 versus control group; ^▲P < 0.01 versus Tan IIA group; ^#P < 0.01 versus ADM group).

Figure 5

Effects of Tan IIA on liver tissue induced hepatotoxicity in histological appearance by ADM in HepG2 tumor-bearing mice (magnification, ×100 and ×400 as indicated). (a) Inflammatory cells infiltration around the central lobular vein. (b) Local hepatocyte necrosis. (c) Hepatic sinusoid congestion (indicated by arrowheads).

Figure 6

The results of microsomal protein, cytochrome b5, cytochrome P450, and erythromycin-N-demethylase (ERND) activity in the liver of HepG2 tumor-bearing mice. (a) The result of liver microsomal protein in tumor-bearing mice. (b) The content of cytochrome b5 in liver microsomes. (c) The content of cytochrome P450 in microsomes. (d) The CYP3A activity in ERND experiment (n = 5, ^∗P < 0.01, ^∗∗P < 0.05 versus control group; ^▲P < 0.01 versus TanIIA group; ^#P < 0.01 versus ADM group.)

Figure 7

Effect of Tan IIA and ADM, respectively, and jointly on protein expression of CYP3A4 in mice liver microsomes. Asterisks indicate a statistically significant difference compared to the model control group (n = 5, ^∗P < 0.01 versus control group; ^▲P < 0.01 versus TanIIA group; ^#P < 0.01, ^##P < 0.05 versus ADM group).

Figure 8

Interactions of Tan IIA and ADM with PXR protein. (a) The 3D structure of PXR from PDB, with an ID of 4NY9 and a resolution of 2.80 Å. It is constituted by 1 peptide chain, which is composed of 290 amino acid residues. An endogenous ligand (yellow) was found within 4NY9. (b) The binding site 1 (green) contains the structure domain of the endogenous ligand after automatic searching. (c) Ten random poses of Tan IIA binding to the endogenous ligand's active site after docking, with one residue involved in the interactions potentially: H-bonds with Ser 247. (d) Ten random poses of Tan IIA binding to site 1 after docking, with the same one residue involved in the interactions potentially: H-bonds with Ser 247. (e) Ten random poses of ADM binding to the endogenous ligand's active site after docking, with six residues involved in the interactions potentially: H-bonds with Lys210, Met243, Ser247, Gln285, Met323, and His407. (f) Ten random poses of ADM binding to site 1 after docking, with five other residues involved in the interactions potentially: H-bonds with Leu206, Leu209, Lys210, Gln285, and His407.